Method of nucleic acid sequence determination

ABSTRACT

Provided are sequencing-by-binding methods of detecting cognate nucleotides using a crippled DNA polymerizing enzyme that possesses the ability to bind the next correct nucleotide downstream of a primer in a template-dependent fashion, but does not possess the activity needed to promote phosphodiester bond formation. Use of the crippled DNA polymerase permits interrogation of one nucleotide at a time, without incorporation of any nucleotide. Labeled nucleotides, such as fluorescently labeled nucleotides, can be used in conjunction with the crippled DNA polymerase to establish cognate nucleotide identity in a rapid manner.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/444,733, filed Jan. 10, 2017; and U.S. Provisional Application No.62/329,489, filed Apr. 29, 2016. The disclosures of these earlierapplications are hereby incorporated by reference in their entireties.

TECHNICAL FIELD

The present invention relates generally to the field of biotechnology.More specifically, the invention concerns nucleic acid sequencingtechnology.

BACKGROUND

Accurate polynucleotide sequence determination is critically importantin many applications. For example, comprehensive definition of anindividual's genetic profile requires that long stretches of chromosomalDNA are determined and compared against databases of known sequences.The results can establish profiles of predispositions orsusceptibilities to provide medical insights. Likewise, tumor profilingalso can require accurate sequence determination to establish theefficacy of a drug treatment regimen before treatment has begun. Aswell, identifying bacterial, viral, or other pathogens from nucleic aciddatabases also can depend on accurate sequencing results.

Stretches of more than one of the same base along a strand of nucleicacid are among the factors confounding accurate sequence determination.These “homopolymer” stretches can be overlooked by some sequencingapproaches, such that a single base will be detected when multiplesactually are present. Some sequencing methods further can experience“phasing” issues that can be exacerbated by the homopolymer stretches.As a consequence of phasing, sequence determination downstream of ahomopolymer stretch can be rendered ambiguous.

Different approaches have been developed to address and resolvehomopolymer sequencing issues. Reversible terminator nucleotides havebeen employed to ensure that only a single nucleotide is enzymaticallyincorporated into a growing primer. While effective, follow-on steps canbe required to remove the chemical terminator moiety from the primerbefore the next round of template-dependent incorporation can occur.Other approaches have involved measuring the amplitude of incorporationsignals using only one species of nucleotide at a time. Unfortunately,this approach imposes limits on the length of sequence that can bedetermined. Thus, there remains a need for new approaches that can beused for sequencing nucleic acids, including accurate sequencing throughhomopolymer stretches within a nucleic acid strand.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an interferometry trace showing the intensity of bindingsignal (vertical axis) as a function of time or reaction progress(horizontal axis). Traces are presented for a control polymerase (“CBT”)and for an engineered polymerase (“TDE”). The CBT polymerase can formternary complexes and incorporate cognate nucleotides. The engineeredTDE polymerase retains the ability to form ternary complexes in thepresence of cognate nucleotide, but cannot incorporate any nucleotide inthe presence of the catalytic Mg²⁺ metal ion. Identities of nucleotidestested in the binding reactions are indicated below the traces (A=dATP;C=dCTP; T=dTTP; G=dGTP). The trace showing the higher binding signal fordCTP, and the lower binding signal for dGTP following the incorporationstep corresponds to the modified TDE polymerase.

SUMMARY OF THE DISCLOSURE

In a first aspect, the disclosure relates to a method of determiningwhether a test nucleotide is the next correct nucleotide having a basecomplementary to the next base in a template strand immediatelydownstream of a primer in a primed template nucleic acid. The methodincludes the step of (a) contacting the primed template nucleic acidwith a first reaction mixture that includes a crippled DNA polymeraseand the test nucleotide. As a result of the contact, if the testnucleotide is the next correct nucleotide, there is formed a complexthat includes the primed template nucleic acid, the crippled DNApolymerase and the test nucleotide. The crippled DNA polymerase used inthe method is substantially unable to catalyze formation ofphosphodiester bonds in the presence of magnesium ions. There also isthe step of (b) measuring binding of the primed template nucleic acid tothe crippled DNA polymerase in the presence of the test nucleotide,without chemical incorporation of the test nucleotide into the primer ofthe primed template nucleic acid. Next, there is the step of (c)determining from the results of step (b) whether the test nucleotide isthe next correct nucleotide. In one embodiment, the first reactionmixture includes a catalytic divalent metal ion. For example, thecatalytic divalent metal ion can be Mg²⁺ ion or Mn²⁺ ion. In each case,the test nucleotide can include an exogenous label. Preferably, theexogenous label of the test nucleotide has a fluorescent moiety, andstep (b) involves measuring a fluorescent signal produced by thefluorescent moiety of the test nucleotide. More preferably, the crippledDNA polymerase has an exogenous label, and step (b) involves detectingthe exogenous label of the crippled DNA polymerase. When this is thecase, the exogenous label of the crippled DNA polymerase can include afluorescent moiety, and step (b) can involve measuring a fluorescentsignal produced by the fluorescent moiety of the crippled DNApolymerase. Generally speaking, and with reference to any of thepreceding embodiments, the primer can include a free 3′ hydroxyl group.Still more generally, and with reference to any of the precedingembodiments, after step (b), there can be the additional step ofreplacing the first reaction mixture with a second reaction mixture thatincludes a second polymerase and a second type of nucleotide, and thenincorporating the second type of nucleotide into the primer of theprimed template nucleic acid. For example, the second type of nucleotidecan be a reversible terminator nucleotide. Yet more generally, and withreference to any of the preceding embodiments, the first reactionmixture can include Mg²⁺ ions, and the primer can include a 3′ hydroxylmoiety. Indeed, the primer of the disclosed method can a 3′ hydroxylmoiety.

In another aspect, the disclosure relates to an isolated mutant DNApolymerase that has the amino acid sequence of SEQ ID NO:12 in place ofSEQ ID NO:13. The mutant DNA polymerase forms ternary complexes withprimed template nucleic acid molecules and cognate nucleotides, and doesnot catalyze phosphodiester bond formation in the presence of Mg²⁺ ion.Preferably, the isolated mutant DNA polymerase includes a reportermoiety attached thereto.

In another aspect, the disclosure relates to an isolated mutant DNApolymerase having the amino acid sequence of SEQ ID NO:14 in place ofSEQ ID NO:15. The mutant DNA polymerase forms ternary complexes withprimed template nucleic acid molecules and cognate nucleotides, and doesnot catalyze phosphodiester bond formation in the presence of Mg²⁺ ion.Preferably, the isolated mutant DNA polymerase further includes areporter moiety attached thereto.

In another aspect, the disclosure relates to an isolated mutant DNApolymerase having the amino acid sequence of SEQ ID NO:3, except forreplacement of aspartate by glutamate at position 355. The mutant DNApolymerase forms ternary complexes with primed template nucleic acidmolecules and cognate nucleotides, and does not catalyze phosphodiesterbond formation in the presence of Mg²⁺ ion. Preferably, the isolatedmutant DNA polymerase further includes an N-terminal sequence of aminoacids having a cysteine residue. More preferably, the cysteine residueis chemically linked to a detectable label.

In another aspect, the disclosure relates to an isolated mutant DNApolymerase having the amino acid sequence of SEQ ID NO:3, except forreplacement of an aspartate residue by a glutamate residue at position532. The mutant DNA polymerase forms ternary complexes with primedtemplate nucleic acid molecules and cognate nucleotides, and does notcatalyze phosphodiester bond formation in the presence of Mg²⁺ ion.Preferably, isolated mutant DNA polymerase further includes anN-terminal sequence of amino acids having a cysteine residue. Morepreferably, the cysteine residue is chemically linked to a detectablelabel.

Further Aspects

Further aspects of the present disclosure are described in the followingnumbered paragraphs.

1. A method of determining whether a test nucleotide is the next correctnucleotide comprising a base complementary to the next base in atemplate strand immediately downstream of a primer in a primed templatenucleic acid, comprising the steps of:

(a) contacting the primed template nucleic acid with a first reactionmixture that comprises a crippled DNA polymerase and the testnucleotide,

whereby, if the test nucleotide is the next correct nucleotide, there isformed a complex comprising the primed template nucleic acid, thecrippled DNA polymerase and the test nucleotide, and

wherein the crippled DNA polymerase is substantially incapable ofmagnesium-catalyzed phosphodiester bond formation;

(b) measuring binding of the primed template nucleic acid to thecrippled DNA polymerase in the presence of the test nucleotide, withoutchemical incorporation of the test nucleotide into the primer of theprimed template nucleic acid; and

(c) determining from the results of step (b) whether the test nucleotideis the next correct nucleotide.

2. The method of paragraph 1, wherein the crippled DNA polymerasecomprises either a polypeptide sequence comprising SEQ ID NO:12, or apolypeptide sequence comprising SEQ ID NO:14.

3. The method of paragraph 1, wherein the crippled DNA polymerasecatalyzes formation of phosphodiester bonds in the presence of divalentmanganese ions, and wherein the first reaction mixture does not containa concentration of divalent manganese ions that promotes formation ofphosphodiester bonds.

4. The method of any one of paragraphs 1-3, wherein the test nucleotidecomprises an exogenous label.

5. The method of paragraph 3, wherein the exogenous label of the testnucleotide comprises a fluorescent moiety, and wherein step (b)comprises measuring a fluorescent signal produced by the fluorescentmoiety of the test nucleotide.

6. The method of any one of paragraphs 1-5, wherein the crippled DNApolymerase comprises an exogenous label, and wherein step (b) comprisesdetecting the exogenous label of the crippled DNA polymerase.

7. The method of paragraph 6, wherein the exogenous label of thecrippled DNA polymerase comprises a fluorescent moiety, and wherein step(b) comprises measuring a fluorescent signal produced by the fluorescentmoiety of the crippled DNA polymerase.

8. The method of any one of paragraphs 1-7, wherein the primer comprisesa free 3′ hydroxyl moiety.

9. The method of any one of paragraphs 1-8, further comprising, afterstep (b), the step of replacing the first reaction mixture with a secondreaction mixture that comprises a second polymerase and a second type ofnucleotide, and then incorporating the second type of nucleotide intothe primer of the primed template nucleic acid.

10. The method of paragraph 9, wherein the second type of nucleotide isa reversible terminator nucleotide that comprises a reversibleterminator moiety, and wherein incorporation of the reversibleterminator nucleotide produces a blocked primed template nucleic acidmolecule.

11. The method of paragraph 10, further comprising the step of removingthe reversible terminator moiety from the blocked primed template nucleiacid molecule to regenerate the primed template nucleic acid molecule.

12. The method of paragraph 10, further comprising repeating steps(a)-(c) using the blocked primed template nucleic acid molecule in placeof the primed template nucleic acid.

13. The method of paragraph 11, further comprising repeating steps(a)-(c).

14. The method of paragraph 1, wherein the polypeptide sequence of thecrippled DNA polymerase is either SEQ ID NO:1 with the exception ofhaving amino acid position 381 substituted by glutamate, or SEQ ID NO:1with the exception of having amino acid position 558 substituted byglutamate.

15. The method of paragraph 1, wherein the polypeptide sequence of thecrippled DNA polymerase is either SEQ ID NO:2 with the exception ofhaving amino acid position 364 substituted by glutamate, or SEQ ID NO:2with the exception of having amino acid position 541 substituted byglutamate.

16. The method of paragraph 1, wherein the polypeptide sequence of thecrippled DNA polymerase is either SEQ ID NO:3 with the exception ofhaving amino acid position 355 substituted by glutamate, or SEQ ID NO:3with the exception of having amino acid position 532 substituted byglutamate.

17. The method of paragraph 1, wherein the first reaction mixturecomprises divalent magnesium ion.

18. The method of any one of paragraphs 1-17, wherein the first reactionmixture comprises Mg²⁺ ions, and wherein the primer comprises a 3′hydroxyl moiety.

19. A kit for identifying a next correct nucleotide to be incorporatedinto a primed template nucleic acid molecule, the kit comprising inpackaged combination of one or more containers:

(a) a crippled DNA polymerase that forms a ternary complex with theprimed template nucleic acid and the next correct nucleotide, but whichis substantially incapable of magnesium-catalyzed phosphodiester bondformation;

(b) four types of deoxyribonucleotide triphosphate molecules; and

(c) four types of reversible terminator nucleotide.

20. The kit of paragraph 19, wherein the four reversible terminatornucleotide are four non-fluorescent reversible terminator nucleotides.

21. The kit of paragraph 19, wherein the crippled DNA polymerasecomprises a detectable label.

22. The kit of paragraph 19, wherein at least one of the four types ofdeoxyribonucleotide triphosphate molecules comprises a detectable label.

23. The kit of paragraph 19, wherein the crippled DNA polymerasecatalyzes phosphodiester bond formation in the presence of manganeseions.

24. The kit of paragraph 19, further comprising a chemical reagent thatremoves reversible terminator moieties from the four types of reversibleterminator nucleotide.

25. The kit of paragraph 19, wherein each of the four different types ofdeoxyribonucleotide triphosphate molecules is incorporable by a DNApolymerase comprising magnesium-dependent polymerase activity.

26. The kit of paragraph 19, further comprising a second DNA polymerasethat incorporates the next correct nucleotide into the primed templatenucleic acid molecule.

27. The kit of paragraph 26, wherein the second DNA polymeraseincorporates one of the four types of reversible terminator nucleotideas the next correct nucleotide.

28. The kit of paragraph 19, further comprising a flow cell.

29. The kit of paragraph 19, wherein the four types ofdeoxyribonucleotide triphosphate molecules comprise dATP, dGTP, dCTP,and either dTTP or dUTP; and wherein the four types of reversibleterminator nucleotides comprise analogs of dATP, dGTP, dCTP, and eitherdTTP or dUTP, each analog comprising a 3′-ONH₂ reversible terminatormoiety.

30. The kit of paragraph 19, wherein the crippled DNA polymerasecomprises either a polypeptide sequence comprising SEQ ID NO:12, or apolypeptide sequence comprising SEQ ID NO:14.

31. The kit of paragraph 30, wherein the polypeptide sequence comprisingSEQ ID NO:12 comprises SEQ ID NO:3 with the exception of having aminoacid position 355 substituted by glutamate.

32. The kit of paragraph 30, wherein the polypeptide sequence comprisingSEQ ID NO:14 comprises SEQ ID NO:3 with the exception of having aminoacid position 532 substituted by glutamate.

33. A mutant DNA polymerase comprising a polypeptide sequence, saidpolypeptide sequence comprising SEQ ID NO:12,

wherein said mutant DNA polymerase forms ternary complexes with primedtemplate nucleic acid molecules and cognate nucleotides, and

wherein said mutant DNA polymerase is substantially incapable ofmagnesium-catalyzed phosphodiester bond formation.

34. The mutant DNA polymerase of paragraph 33, wherein the crippled DNApolymerase catalyzes formation of phosphodiester bonds in the presenceof divalent manganese ions.

35. The mutant DNA polymerase of paragraph 33, wherein the polypeptidesequence of the mutant DNA polymerase is selected from the groupconsisting of:

SEQ ID NO:1 with the exception of having amino acid position 381substituted by glutamate,

SEQ ID NO:2 with the exception of having amino acid position 364substituted by glutamate, and

SEQ ID NO:3 with the exception of having amino acid position 355substituted by glutamate.

36. The mutant DNA polymerase of paragraph 35, further comprising areporter moiety attached thereto.

37. A mutant DNA polymerase comprising a polypeptide sequence, saidpolypeptide sequence comprising SEQ NO:14,

wherein said mutant DNA polymerase forms ternary complexes with primedtemplate nucleic acid molecules and cognate nucleotides, and

wherein said mutant DNA polymerase is substantially incapable ofmagnesium-catalyzed phosphodiester bond formation.

38. The mutant DNA polymerase of paragraph 37, wherein the polypeptidesequence of the mutant DNA polymerase is selected from the groupconsisting of:

SEQ ID NO:1 with the exception of having amino acid position 558substituted by glutamate,

SEQ ID NO:2 with the exception of having amino acid position 541substituted by glutamate, and

SEQ ID NO:3 with the exception of having amino acid position 532substituted by glutamate.

39. The isolated mutant DNA polymerase of paragraph 38, furthercomprising a reporter moiety attached thereto.

DETAILED DESCRIPTION

The following description relates to a sequencing-by-binding (SBB)technique that relies on use of a mutant, non-catalytic DNA polymerase(a “crippled” polymerase). Crippled DNA polymerase enzymes useful forpracticing the technique are substantially unable to catalyzemagnesium-dependent formation of a phosphodiester bond between a primerstrand of a primed template nucleic acid, and an incoming next correctnucleotide. Although being without this catalytic activity, the mutantenzyme retains the ability to discriminate cognate from non-cognatenucleotides. In some embodiments, the mutant polymerase is unable tobind the divalent cation ordinarily needed for catalytic function.Although the following description exemplifies useful aspects ofcrippled polymerase in the context of SBB, it will be understood thatthe polymerase can have other uses including, but not limited to,identifying individual nucleotides in a nucleic acid (e.g., detectingsingle nucleotide polymorphisms) or binding to particular sequences toprovide polymerase-mediated affinity separation of the nucleic acidsthat bear the sequences. Advantageously, use of crippled DNA polymerasesovercome problems associated with undesired incorporation of residualnucleotides that may remain following wash steps, or undesiredincorporation resulting from incomplete inhibition that can be achievedusing non-catalytic metal ions that inhibit polymerase activity. Thus,use of the crippled DNA polymerase can reduce sequencing artifactsassociated with undesired incorporation of nucleotides.

Briefly, formation of a stable complex that includes a primed templatenucleic acid, a cognate nucleotide, and a crippled DNA polymerase, canbe detected even in the presence of catalytic divalent cations. Theprimer need not have an extension blocking group and the nucleotide neednot have moieties that inhibit incorporation into the primer. Aftercognate nucleotide for a particular position has been identified, or atleast monitoring or measuring information needed to make theidentification has been gathered, the primed template nucleic acid canbe contacted with a different reaction mixture that includes second DNApolymerase instead of the crippled DNA polymerase. The second DNApolymerase, when further provided with a cognate nucleotide andappropriate divalent cation, will be capable of joining the cognatenucleotide to the primer at the position of a free 3′ hydroxyl group. Ifa reversible terminator nucleotide is employed in this step, then only asingle incorporation will occur.

Advantageously, the technique can be practiced using various types ofnucleotides, including native (e.g., unlabeled) nucleotides, nucleotideswith detectable labels (e.g., fluorescent or other optically detectablelabels), or labeled or unlabeled nucleotide analogs (e.g., modifiednucleotides containing reversible terminator moieties). Further, thetechnique provides controlled reaction conditions, unambiguousdetermination of sequence, low overall cost of reagents, and lowinstrument cost.

The disclosed technique can be applied to binding reactions used fordetermining the identity of the next base of a primed template nucleicacid by any means and for any reason. The technique can be used tomonitor specific binding of a DNA polymerase and the next correctnucleotide (e.g., a dNTP) complementary to a primed template nucleicacid, and to distinguish specific binding from nonspecific binding. Thetechnique may be applied to single nucleotide determination (e.g., SNPdetermination), or alternatively to more extensive nucleic acidsequencing procedures employing iterative cycles that identify onenucleotide at a time. For example, the methods provided herein can beused in connection with sequencing-by-binding procedures, as describedin the commonly owned U.S. patent application identified by Ser. No.14/805,381 (published as U.S. Pat. App. Pub. No. 2017/0022553 A1), thedisclosure of which is incorporated by reference herein in its entirety.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. For clarity, the following specific terms have the specifiedmeanings. Other terms are defined in other sections herein.

The singular forms “a” “an” and “the” include plural referents unlessthe context clearly dictates otherwise. Approximating language, as usedin the description and claims, may be applied to modify any quantitativerepresentation that could permissibly vary without resulting in a changein the basic function to which it is related. Accordingly, a valuemodified by a term such as “about” is not to be limited to the precisevalue specified. Unless otherwise indicated, all numbers expressingquantities of ingredients, properties such as molecular weight, reactionconditions, so forth used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the compositions, apparatus, or methods of thepresent disclosure. At the very least, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

As used herein, “sequencing-by-binding” refers to a sequencing techniquewherein specific binding of a polymerase to a primed template nucleicacid is used for identifying the next correct nucleotide to beincorporated into the primer strand of the primed template nucleic acid.The specific binding interaction precedes chemical incorporation of thenucleotide into the primer strand, and so identification of the nextcorrect nucleotide can take place either without or before incorporationof the next correct nucleotide.

As used herein, “nucleic acid” or “oligonucleotide” or “polynucleotide”or grammatical equivalents used herein means at least two nucleotidescovalently linked together. Thus, a “nucleic acid” is a polynucleotide,such as DNA, RNA, or any combination thereof, that can be acted upon bya polymerizing enzyme during nucleic acid synthesis. The term “nucleicacid” includes single-, double-, or multiple-stranded DNA, RNA andanalogs (derivatives) thereof. Double-stranded nucleic acidsadvantageously can minimize secondary structures that may hinder nucleicacid synthesis. A double stranded nucleic acid may possess a nick or asingle-stranded gap.

As used herein, a “template nucleic acid” is a nucleic acid to bedetected or sequenced using any sequencing method disclosed herein.

As used herein, a “primed template nucleic acid” (or alternatively,“primed template nucleic acid molecule”) is a template nucleic acidprimed with (i.e., hybridized to) a primer, wherein the primer is anoligonucleotide having a 3′-end with a sequence complementary to aportion of the template nucleic acid. The primer can optionally have afree 5′-end (e.g., the primer being noncovalently associated with thetemplate) or the primer can be continuous with the template (e.g., via ahairpin structure). The primed template nucleic acid includes thecomplementary primer and the template nucleic acid to which it is bound.Unless explicitly stated, a primed template nucleic acid can have eithera 3′-end that is extendible by a polymerase, or a 3′-end that is blockedfrom extension.

As used herein, a “blocked primed template nucleic acid” (oralternatively, “blocked primed template nucleic acid molecule”) is aprimed template nucleic acid modified to preclude or preventphosphodiester bond formation at the 3′-end of the primer. Blocking maybe accomplished, for example, by chemical modification with a blockinggroup at either the 3′ or 2′ position of the five-carbon sugar at the 3′terminus of the primer. Alternatively, or in addition, chemicalmodifications that preclude or prevent phosphodiester bond formation mayalso be made to the nitrogenous base of a nucleotide. Reversibleterminator nucleotide analogs including each of these types of blockinggroups will be familiar to those having an ordinary level of skill inthe art. Incorporation of these analogs at the 3′ terminus of a primerof a primed template nucleic acid molecule results in a blocked primedtemplate nucleic acid molecule. The blocked primed template nucleic acidincludes the complementary primer, blocked from extension at its 3′-end,and the template nucleic acid to which it is bound.

As used herein, a “nucleotide” is a molecule that includes a nitrogenousbase, a five-carbon sugar (ribose or deoxyribose), and at least onephosphate group. The term embraces, but is not limited to,ribonucleotides, deoxyribonucleotides, nucleotides modified to includeexogenous labels or reversible terminators, and nucleotide analogs.

As used herein, a “native” nucleotide refers to a naturally occurringnucleotide that does not include an exogenous label (e.g., a fluorescentdye, or other label) or chemical modification such as may characterize anucleotide analog. Examples of native nucleotides useful for carryingout the sequencing-by-binding procedures described herein include: dATP(2′-deoxyadenosine-5′-triphosphate); dGTP(2′-deoxyguanosine-5′-triphosphate); dCTP(2′-deoxycytidine-5′-triphosphate); dTTP(2′-deoxythymidine-5′-triphosphate); and dUTP(2′-deoxyuridine-5′-triphosphate).

As used herein, a “nucleotide analog” has one or more modifications,such as chemical moieties, which replace, remove and/or modify any ofthe components (e.g., nitrogenous base, five-carbon sugar, or phosphategroup(s)) of a native nucleotide. Nucleotide analogs may be eitherincorporable or non-incorporable by a polymerase in a nucleic acidpolymerization reaction. Optionally, the 3′-OH group of a nucleotideanalog is modified with a moiety. The moiety may be a 3′ reversible orirreversible terminator of polymerase extension. The base of anucleotide may be any of adenine, cytosine, guanine, thymine, or uracil,or analogs thereof. Optionally, a nucleotide has an inosine, xanthine,hypoxanthine, isocytosine, isoguanine, nitropyrrole (including3-nitropyrrole) or nitroindole (including 5-nitroindole) base.Nucleotides may include, but are not limited to, ATP, UTP, CTP, GTP,ADP, UDP, CDP, GDP, AMP, UMP, CMP, GMP, dATP, dTTP, dUTP, dCTP, dGTP,dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Nucleotides may alsocontain terminating inhibitors of DNA polymerase, dideoxynucleotides or2′,3′ dideoxynucleotides, which are abbreviated as ddNTPs (ddGTP, ddATP,ddTTP, ddUTP and ddCTP).

As used herein, the “next template nucleotide” (or the “next templatebase”) refers to the next nucleotide (or base) in a template nucleicacid that is located immediately downstream of the 3′-end of ahybridized primer. In other words, the next template nucleotide islocated immediately 5′ of the base in the template that is hybridized tothe 3′ end of the primer.

As used herein, a “blocking moiety,” when used with reference to anucleotide analog, is a part of the nucleotide that inhibits or preventsthe nucleotide from forming a covalent linkage to a second nucleotide(e.g., via the 3′-OH of a primer nucleotide) during the incorporationstep of a nucleic acid polymerization reaction. The blocking moiety of a“reversible terminator” nucleotide can be removed from the nucleotideanalog to allow for nucleotide incorporation. Such a blocking moiety isreferred to herein as a “reversible terminator moiety.” Exemplaryreversible terminator moieties are set forth in U.S. Pat. Nos.7,427,673; 7,414,116; and 7,057,026 and PCT publications WO 91/06678 andWO 07/123744, each of which is incorporated by reference.

As used herein, a “test nucleotide” is a nucleotide being investigatedfor its ability to participate in formation of a ternary complex thatfurther includes a primed template nucleic acid and a polymerase.

As used herein, “polymerase” is a generic term for a nucleic acidsynthesizing enzyme, including but not limited to, DNA polymerase, RNApolymerase, reverse transcriptase, primase and transferase. Typically,the polymerase includes one or more active sites at which nucleotidebinding and/or catalysis of nucleotide polymerization may occur. Thepolymerase may catalyze the polymerization of nucleotides to the 3′-endof a primer bound to its complementary nucleic acid strand. For example,a polymerase can catalyze the addition of a next correct nucleotide tothe 3′ oxygen of the primer via a phosphodiester bond, therebychemically incorporating the nucleotide into the primer. Optionally, thepolymerase used in the provided methods is a processive polymerase.Optionally, the polymerase used in the provided methods is adistributive polymerase. Optionally, a polymerase need not be capable ofnucleotide incorporation under one or more conditions used in a methodset forth herein. For example, a mutant polymerase may be capable offorming a ternary complex but incapable of catalyzing nucleotideincorporation.

As used herein, a “salt providing monovalent cation” is an ioniccompound that dissociates in aqueous solution to produce cations havinga single positive charge. For example, the cations can be metal cationswhere the oxidation state is +1.

As used herein, “a glutamate salt” is an ionic compound that dissociatesin aqueous solution to produce glutamate anions.

As used herein, “biphasic” refers to a two-stage process wherein aprimed template nucleic acid is contacted with a polymerase and a testnucleotide. The first phase of the process involves contacting theprimed template nucleic acid with a polymerase in the presence of asub-saturating level of nucleotide(s), or even in the absence ofnucleotides. The term “sub-saturating,” when used in reference to ligandthat binds to a receptor (e.g., a nucleotide that binds to apolymerase), refers to a concentration of the ligand that is below thatrequired to result in at least 90% of the receptors being bound to theligand at equilibrium. For example, a sub-saturating amount ofnucleotide can yield at least 90%, 95%, 99% or more polymerases beingbound to the nucleotide. The second phase of the process involvescontacting the primed template nucleic acid from the first phase with apolymerase in the presence of a higher concentration of nucleotide(s)than used in the first phase, where the higher concentration issufficient to yield maximal ternary complex formation when a nucleotidein the reaction is the next correct nucleotide.

As used herein, “providing” a template, a primer, a primed templatenucleic acid, or a blocked primed template nucleic acid refers to thepreparation and delivery of one or many nucleic acid polymers, forexample to a reaction mixture or reaction chamber.

As used herein, “monitoring” (or sometimes “measuring”) refers to aprocess of detecting a measurable interaction or binding between twomolecular species. For example, monitoring may involve detectingmeasurable interactions between a polymerase and primed template nucleicacid, typically at various points throughout a procedure. Monitoring canbe intermittent (e.g., periodic) or continuous (e.g., withoutinterruption), and can involve acquisition of quantitative results.Monitoring can be carried out by detecting multiple signals over aperiod of time during a binding event or, alternatively, by detectingsignal(s) at a single time point during or after a binding event.

As used herein, “contacting” refers to the mixing together of reagents(e.g., mixing an immobilized template nucleic acid and either a bufferedsolution that includes a polymerase, or the combination of a polymeraseand a test nucleotide) so that a physical binding reaction or a chemicalreaction may take place.

As used herein, “incorporating” or “chemically incorporating,” when usedin reference to a primed template and nucleotide, refers to the processof joining a cognate nucleotide to a primer by formation of aphosphodiester bond.

As used herein, “extension” refers to the process after anoligonucleotide primer and a template nucleic acid have annealed to oneanother, wherein a polymerase enzyme catalyzes addition of one or morenucleotides at the 3′-end of the primer. A nucleotide that is added to anucleic acid by extension is said to be “incorporated” into the nucleicacid. Accordingly, the term “incorporating” can be used to refer to theprocess of joining a nucleotide to the 3′-end of a primer by formationof a phosphodiester bond.

As used herein, a “binary complex” is an intermolecular associationbetween a polymerase and a primed template nucleic acid (or blockedprimed template nucleic acid), where the complex does not include anucleotide molecule such as the next correct nucleotide.

As used herein, a “ternary complex” is an intermolecular associationbetween a polymerase, a primed template nucleic acid (or blocked primedtemplate nucleic acid), and the next correct nucleotide positionedimmediately downstream of the primer and complementary to the templatestrand of the primed template nucleic acid or the blocked primedtemplate nucleic acid. The primed template nucleic acid can include, forexample, a primer with a free 3′-OH or a blocked primer (e.g., a primerwith a chemical modification on the base or the sugar moiety of the 3′terminal nucleotide, where the modification precludes enzymaticphosphodiester bond formation). The term “stabilized ternary complex”means a ternary complex having promoted or prolonged existence or aternary complex for which disruption has been inhibited. Generally,stabilization of the ternary complex prevents covalent incorporation ofthe nucleotide component of the ternary complex into the primed nucleicacid component of the ternary complex.

As used herein, a “catalytic metal ion” refers to a metal ion thatfacilitates phosphodiester bond formation between the 3′-OH of a nucleicacid (e.g., a primer) and the phosphate of an incoming nucleotide by apolymerase. A “divalent catalytic metal cation” is a catalytic metal ionhaving a valence of two. Catalytic metal ions can be present atconcentrations necessary to stabilize formation of a complex between apolymerase, a nucleotide, and a primed template nucleic acid, referredto as non-catalytic concentrations of a metal ion. Catalyticconcentrations of a metal ion refer to the amount of a metal ionsufficient for polymerases to catalyze the reaction between the 3′-OHgroup of a nucleic acid (e.g., a primer) and the phosphate group of anincoming nucleotide.

As used herein, a “non-catalytic metal ion” refers to a metal ion that,when in the presence of a polymerase enzyme, does not facilitatephosphodiester bond formation needed for chemical incorporation of anucleotide into a primer. Typically, the non-catalytic metal ion is acation. A non-catalytic metal ion may inhibit phosphodiester bondformation by a polymerase, and so may stabilize a ternary complex bypreventing nucleotide incorporation. Non-catalytic metal ions mayinteract with polymerases, for example, via competitive binding comparedto catalytic metal ions. A “divalent non-catalytic metal ion” is anon-catalytic metal ion having a valence of two. Examples of divalentnon-catalytic metal ions include, but are not limited to, Ca²⁺, Zn²⁺,Co²⁺, Ni²⁺, and Sr²⁺. The trivalent Eu³⁺ and Tb³⁺ ions are non-catalyticmetal ions having a valence of three.

As used herein an “exogenous label” refers to a detectable chemicalmoiety that has been added to a sequencing reagent, such as a nucleotideor a polymerase (e.g., a DNA polymerase). While a native dNTP may have acharacteristic limited fluorescence profile, the native dNTP does notinclude any added colorimetric or fluorescent moiety. Conversely, a dATP(2′-deoxyadenosine-5′-triphosphate) molecule modified to include achemical linker and fluorescent moiety attached to the gamma phosphatewould be said to include an exogenous label because the attachedchemical components are not ordinarily a part of the nucleotide. Ofcourse, chemical modifications to add detectable labels to nucleotidebases also would be considered exogenous labels. Likewise, a DNApolymerase modified to include a conformationally sensitive fluorescentdye that changes its properties upon nucleotide binding also would besaid to include an exogenous label because the label is not ordinarily apart of the polymerase.

As used herein, “unlabeled” refers to a molecular species free of addedor exogenous label(s) or tag(s). Of course, unlabeled nucleotides willnot include either of an exogenous fluorescent label, or an exogenousRaman scattering tag. A native nucleotide is another example of anunlabeled molecular species. An unlabeled molecular species can excludeone or more of the labels set forth herein or otherwise known in the artrelevant to nucleic acid sequencing or analytical biochemistry.

As used herein, the term “solid support” refers to a rigid substratethat is insoluble in aqueous liquid. The substrate can be non-porous orporous. The substrate can optionally be capable of taking up a liquid(e.g., due to porosity) but will typically be sufficiently rigid thatthe substrate does not swell substantially when taking up the liquid anddoes not contract substantially when the liquid is removed by drying. Anonporous solid support is generally impermeable to liquids or gases.Exemplary solid supports include, but are not limited to, glass andmodified or functionalized glass, plastics (including acrylics,polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™,cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor,silica or silica-based materials including silicon and modified silicon,carbon, metals, inorganic glasses, optical fiber bundles, and polymers.

As used herein, a “native DNA polymerase” is a modified or unmodifiedDNA polymerase enzyme that possesses the ability to discriminate cognatenucleotide from a non-cognate nucleotide, and to incorporate the cognatenucleotide into the growing primer strand, by forming a phosphodiesterbond joining the two species when also provided with a catalyticdivalent metal ion (e.g., Mg²⁺ or Mn²⁺).

As used herein, a “crippled DNA polymerase” is a mutated version of aDNA polymerizing enzyme, where the mutant is capable of discriminatingthe next correct nucleotide from an incorrect nucleotide (e.g., beingable to form a template-dependent complex with a primed template nucleicacid and cognate nucleotide), but incapable of catalyzing phosphodiesterbond formation between the primer and the cognate nucleotide. Forexample, one or more amino acid positions of a native DNA polymerase canbe modified (e.g., by site-directed mutagenesis of the polynucleotideencoding same and/or by chemical modification of one or more sites inthe polynucleotide) to create the crippled DNA polymerase.

As used herein, a “kit” is a packaged unit containing one or morecomponents that can be used for performing detection and/or sequencingreactions using a crippled DNA polymerase, as disclosed herein. Typicalkits may include packaged combinations, in one or more containers orvials, of reagents to be used in the procedure.

Sequencing-by-Binding

Described herein are polymerase-based, nucleic acidsequencing-by-binding (SBB) reactions, wherein a crippled DNA polymeraseundergoes a conformational transition upon encountering a primedtemplate nucleic acid molecule and the next correct nucleotide. Thecrippled DNA polymerase is unable to catalyze phosphodiester bondformation, and so lingers in the complex referred to herein as the“ternary” complex. Detection of the ternary complex by any approach is asurrogate for detecting the cognate nucleotide.

The disclosed approach shares many features in common with conventionalSBB techniques (disclosed in the commonly owned U.S. patent applicationidentified by Ser. No. 14/805,381), which will be described below ingeneral terms. Of course, any reference to magnesium-based catalysis bythe same enzyme used for the examination step do not apply to thecrippled DNA polymerase.

In one step, the polymerase binds to a primed template nucleic acid toform a binary complex, also referred to herein as the pre-insertionconformation. In a subsequent step, an incoming nucleotide is bound andthe polymerase fingers close, forming a pre-chemistry conformationincluding a polymerase, primed template nucleic acid and nucleotide;wherein the bound nucleotide has not been incorporated. This step, alsoreferred to herein as the “examination” step, may be followed by achemical step wherein a phosphodiester bond is formed with concomitantpyrophosphate cleavage from the nucleotide (i.e., nucleotideincorporation). The polymerase, primed template nucleic acid and newlyincorporated nucleotide produce a post-chemistry, pre-translationconformation. As both the pre-chemistry conformation and thepre-translocation conformation include a polymerase, primed templatenucleic acid and nucleotide, wherein the polymerase is in a closedstate, either conformation may be referred to herein as a closed-complexor a closed ternary complex. In the closed pre-insertion state, divalentcatalytic metal ions, such as Mg²⁺ mediate a rapid chemical reactioninvolving nucleophilic displacement of a pyrophosphate (PPi) by the 3′hydroxyl of the primer. The polymerase returns to an open state upon therelease of PPi, the post-translocation step, and translocation initiatesthe next round of reaction. While a closed-complex can form in theabsence of divalent catalytic metal ions (e.g., Mg²), the polymerase ofthe closed complex is proficient in chemical addition of nucleotide inthe presence of the divalent metal ions when provided with anappropriate substrate having an available 3′ hydroxyl group. Low ordeficient levels of catalytic metal ions, such as Mg²⁺, lead tonon-covalent (e.g., physical) sequestration of the next correctnucleotide in a closed-complex. This closed-complex may be referred toas a stabilized or trapped closed-complex. In any reaction stepdescribed above, the polymerase configuration and/or interaction with anucleic acid may be monitored during an examination step to identify thenext correct base in the template nucleic acid sequence. Before or afterincorporation, reaction conditions can be changed to disengage thepolymerase from the primed template nucleic acid, and changed again toremove from the local environment any reagents that inhibit polymerasebinding.

Generally speaking, the SBB procedure includes an “examination” stepthat identifies the next template base, and optionally an“incorporation” step that adds one or more complementary nucleotides tothe 3′-end of the primer component of the primed template nucleic acid.Identity of the next correct nucleotide to be added is determined eitherwithout, or before chemical linkage of that nucleotide to the 3′-end ofthe primer through a covalent bond. The examination step can involveproviding a primed template nucleic acid to be used in the procedure,and contacting the primed template nucleic acid with a polymerase enzyme(e.g., a DNA polymerase) and one or more test nucleotides beinginvestigated as the possible next correct nucleotide. Further, there isa step that involves monitoring or measuring the interaction between thepolymerase and the primed template nucleic acid in the presence of thetest nucleotides. Optionally, the interaction can take place in thepresence of stabilizers, whereby the polymerase-nucleic acid interactionis stabilized in the presence of the next correct nucleotide. Again, theexamination step identifies or determines the identity of the nextcorrect nucleotide without requiring incorporation of that nucleotide.Stated differently, identity of the next correct nucleotide can beestablished without chemical incorporation of the nucleotide into theprimer when one or more cycles of examination is carried out usinglabeled or unlabeled nucleotides.

Whereas methods involving a single template nucleic acid molecule may bedescribed for convenience, these methods are exemplary. The sequencingmethods provided herein readily encompass a plurality of templatenucleic acids, wherein the plurality of nucleic acids may be clonallyamplified copies of a single nucleic acid, or disparate nucleic acids,including combinations, such as populations of disparate nucleic acidsthat are clonally amplified. Thus, such sequencing methods are fullydisclosed herein.

The Examination Step

An examination step according to the technique described hereintypically includes the following substeps: (1) providing a primedtemplate nucleic acid (i.e., a template nucleic acid molecule hybridizedwith a primer that optionally may be blocked from extension at its3′-end); (2) contacting the primed template nucleic acid with a reactionmixture that includes a crippled DNA polymerase and at least onenucleotide; (3) monitoring the interaction of the crippled DNApolymerase with the primed template nucleic acid molecule in thepresence of the nucleotide(s) and without chemical incorporation of anynucleotide into the primed template nucleic acid; and (4) identifyingthe next base in the template nucleic acid (i.e., the next correctnucleotide) using the monitored interaction. Optionally, the primedtemplate nucleic acid molecule can be contacted initially with thecrippled DNA polymerase in the absence of nucleotide(s) beforecontacting any nucleotide. The primer of the primed template nucleicacid can be an extendible primer. The primed template nucleic acid, thecrippled DNA polymerase and the test nucleotide are capable of forming aternary complex when the base of the test nucleotide is complementary tothe next base of the primed template nucleic acid molecule. The primedtemplate nucleic acid and the crippled DNA polymerase are capable offorming a binary complex when the base of the test nucleotide is notcomplementary to the next base of the primed template nucleic acidmolecule. Optionally, the contacting occurs under conditions that favorformation of the ternary complex over formation of the binary complex.The identifying step can include identifying the base of the nucleotidethat is complementary to the next base of the primed template nucleicacid. Optionally, this includes contacting ternary complexes with one ormore wash solutions having different nucleotide compositions that permitternary complexes to be selectively maintained or dissociated.

All of these steps can be repeated one or more times to obtain extensivesequence information. For example, ternary complexes can be formedinitially by contacting a primed template nucleic acid (optionallyincluding a blocked 3′-end) with a crippled DNA polymerase (optionallylabeled with an exogenous label) and one or more nucleotides (optionallyincluding one or more exogenous labels). Buffer conditions can bechanged such that ternary complexes are contacted with a wash solutionthat includes only a subset of nucleotides used for forming the ternarycomplex. Optionally, this buffer includes the same crippled DNApolymerase used to form the ternary complex. Monitoring interaction ofthe crippled DNA polymerase and/or nucleotide in the ternary complex canbe carried out to determine whether the ternary complex remains stable(thereby indicating that one of the nucleotides in the wash buffercorresponds to the cognate nucleotide) or becomes destabilized (therebyindicating that the buffer no longer contains the cognate nucleotide).The wash steps can be repeated until the ternary complex becomesdestabilized by progressively omitting one nucleotide that was presentduring the preceding wash cycle. Optionally, a cognate nucleotide can beincorporated by a second DNA polymerase following one or a plurality ofreagent exchanges.

All of these steps can be repeated one or more times to obtain extensivesequence information. For example, the contacting and monitoring stepscan be repeated one or more times. Optionally, the contacting andmonitoring steps are repeated using a reaction mixture that includes thecrippled DNA polymerase and a first test nucleotide. Optionally, thecontacting and monitoring steps are repeated using a reaction mixturethat includes the crippled DNA polymerase and a second nucleotide.Optionally, the contacting and monitoring steps are repeated using areaction mixture that includes the crippled DNA polymerase and a thirdnucleotide. Optionally, the contacting and monitoring steps are repeatedusing a reaction mixture that includes the crippled DNA polymerase and afourth nucleotide.

In the sequencing methods provided herein, the reaction mixture used forforming ternary complexes, that includes the crippled DNA polymerase andat least one test nucleotide, can include at least 1, 2, 3, or 4 typesof nucleotide molecules (e.g., either labeled or unlabeled nucleotides).Alternatively or additionally, the reaction mixture can include at most4, 3, 2 or 1 types of nucleotide molecules. Optionally, the nucleotidesare native nucleotides selected from dATP, dTTP, dCTP, and dGTP.Optionally, the reaction mixture includes one or more triphosphatenucleotides and one or more diphosphate nucleotides. Optionally, aclosed-complex is formed between the primed template nucleic acid, thecrippled DNA polymerase, and one of four nucleotide molecules includedin the reaction mixture.

In a particular example of the provided method, the primed templatenucleic acid (optionally blocked at its 3′-end) is contacted with areaction mixture that includes polymerase with one or more nucleotides.A ternary complex will form if one or more of the nucleotides is acognate nucleotide for the position being interrogated.

In another particular example of the provided method, the primedtemplate nucleic acid (optionally blocked at its 3′-end) is initiallycontacted with a reaction mixture that includes the crippled DNApolymerase without added test nucleotide. Thereafter, the primedtemplate nucleic acid is contacted with a reaction mixture that includesthe crippled DNA polymerase and one or more test nucleotides that mayparticipate in ternary complex formation. Thereafter, the optionallyblocked primed template nucleic acid is contacted with a reactionmixture that includes polymerase and one fewer nucleotide than thepreceding reaction mixture. Monitoring maintenance or destabilization ofany ternary complex can take place continuously, or after each reactionmixture change.

Since nucleotide incorporation does not take place during theexamination step, a separate incorporation step may be performed afterdetermining the identity of the next correct nucleotide, or at leastacquiring the results necessary to make the determination. The separateincorporation step may be accomplished without the need for monitoring,as the cognate nucleotide has already been identified during theexamination step. A reversibly terminated nucleotide may also be used toprevent the addition of subsequent nucleotides. The SBB method allowsfor controlled determination of a template nucleic acid base with orwithout the use of labeled nucleotides, as the interaction between thecrippled DNA polymerase and template nucleic acid can be monitored withor without a label on the nucleotide. To be clear, however, the use of alabeled nucleotide (e.g., a fluorescent nucleotide) is optional whenperforming the presently disclosed procedure to allow for fluorescentdetection of bound nucleotide.

In the sequencing methods provided herein, the test nucleotide (e.g., atleast one test nucleotide) can include a 3′ hydroxyl group, or ablocking moiety that prevents phosphodiester bond formation at the3′-end of the primer. A 3′ terminator moiety or a 2′ terminator moietymay be either a reversible terminator or an irreversible terminator.Optionally, the reversible terminator of the at least one nucleotidemolecule is replaced or removed at some point after the examination stepthat employed the test nucleotide that included the reversibleterminator.

Contacting Steps

Contacting of the primed template nucleic acid molecule with reactionmixtures that include the crippled DNA polymerase and one or more testnucleotide molecules can occur under conditions that stabilize formationof the ternary complex and/or destabilize formation of the binarycomplex. Optionally, the reaction mixture includes potassium glutamate.Optionally, the conditions that stabilize formation of the ternarycomplex include contacting the primed template nucleic acid with astabilizing agent. Optionally, the reaction mixture includes astabilizing agent. The stabilizing agent can be one or morenon-catalytic metal ions that inhibit polymerase-mediated incorporation.Exemplary non-catalytic metal ions useful for this purpose includestrontium ion, tin ion, nickel ion, and europium ion. For example, thereaction mixture of the examination step that includes the primedtemplate nucleic acid, the polymerase, and the test nucleotide also mayinclude from 0.01 mM to 30 mM strontium chloride as a stabilizing agent.

Alternatively, and particularly when using a blocked primed templatenucleic acid to form a ternary complex in the examination step, reactionmixtures used for conducting examination and monitoring steps optionallycan include catalytic metal ions (e.g., Mg²⁺ or Mn²⁺). Concentrations ofthe catalytic metal ions needed to support polymerization activity whenusing unmodified (i.e., not 3′ blocked) primers will be familiar tothose having an ordinary level of skill in the art.

In certain embodiments, the primed template nucleic acid is immobilizedto the surface of a solid support. The immobilization may employ eithera covalent or a noncovalent bond between one or the other, or even bothstrands of the primed template nucleic acid and the solid support. Forexample, when the template and primer strands of the primed templatenucleic acid are different molecules, the template strand can beimmobilized, for example via its 5′-end. What is necessary, however, isthat the 3′ terminus of the primer is available for interacting with thepolymerase.

When the primed template nucleic acid is immobilized to a solid support,there are alternatives for how the contacting steps are performed. Forexample, the solid support can be physically transferred betweendifferent vessels (e.g., individual wells of a multiwell plate)containing different reagent solutions. This is convenientlyaccomplished using an automated or robotic instrument. In anotherexample, the primed template nucleic acid is immobilized to a solidsupport inside a flow cell or chamber. In this instance, differentcontacting steps can be executed by controlled flow of different liquidreagents through the chamber, or across the immobilized primed templatenucleic acid.

The Monitoring Step

Monitoring or measuring the interaction of the crippled DNA polymerasewith the primed template nucleic acid molecule in the presence of anucleotide molecule may be accomplished in many different ways. Forexample, monitoring can include measuring association kinetics for theinteraction between the primed template nucleic acid, the crippled DNApolymerase, and a nucleotide. Monitoring the interaction of the crippledDNA polymerase with the primed template nucleic acid molecule in thepresence of a nucleotide molecule can include measuring equilibriumbinding constants between the crippled DNA polymerase and primedtemplate nucleic acid molecule (i.e., equilibrium binding constants ofpolymerase to the template nucleic acid in the presence of anucleotide). Thus, for example, the monitoring includes measuring theequilibrium binding constant of the crippled DNA polymerase to theprimed template nucleic acid in the presence of a nucleotide. Monitoringthe interaction of the crippled DNA polymerase with the primed templatenucleic acid molecule in the presence of a nucleotide molecule includesmeasuring dissociation kinetics of the polymerase from the primedtemplate nucleic acid in the presence of any one of the fournucleotides. Optionally, monitoring the interaction of the crippled DNApolymerase with the primed template nucleic acid molecule in thepresence of a nucleotide molecule includes measuring kinetics of thedissociation of the closed-complex (i.e., dissociation of the primedtemplate nucleic acid, the polymerase, and the nucleotide). Optionally,the measured association kinetics differ depending on the identity ofthe nucleotide molecule. Optionally, the crippled DNA polymerase has adifferent affinity for each type of nucleotide employed. Optionally, thecrippled DNA polymerase has a different dissociation constant for eachtype of nucleotide in each type of closed-complex. Association,equilibrium and dissociation kinetics are known and can be readilydetermined by one in the art. See, for example, Markiewicz et al.,Nucleic Acids Research 40(16):7975-84 (2012); Xia et al., J. Am. Chem.Soc. 135(1):193-202 (2013); Brown et al., J. Nucleic Acids, Article ID871939, 11 pages (2010); Washington, et al., Mol. Cell. Biol.24(2):936-43 (2004); Walsh and Beuning, J. Nucleic Acids, Article ID530963, 17 pages (2012); and Roettger, et al., Biochemistry47(37):9718-9727 (2008), which are incorporated by reference herein intheir entireties.

The monitoring step can include monitoring the steady state interactionof the polymerase with the primed template nucleic acid in the presenceof a first nucleotide, without chemical incorporation of the firstnucleotide into the primer of the primed template nucleic acid.Optionally, monitoring includes monitoring the dissociation of thepolymerase from the primed template nucleic acid in the presence of afirst nucleotide, without chemical incorporation of the first nucleotideinto the primer of the primed template nucleic acid. Optionally,monitoring includes monitoring the association of the polymerase withthe primed template nucleic acid in the presence of the firstnucleotide, without chemical incorporation of the first nucleotide intothe primer of the primed template nucleic acid. Again, test nucleotidesin these procedures may be native nucleotides (i.e., unlabeled), labelednucleotides (e.g., fluorescently labeled nucleotides), or nucleotideanalogs (e.g., nucleotides modified to include reversible orirreversible terminator moieties).

Since the crippled DNA polymerase is substantially unable to catalyzephosphodiester bond formation, it is optional to include a catalyticmetal ion in the examination reaction mixture. Of course, the absence ofa catalytic metal ion in the reaction mixture, or the absence of acatalytic metal ion in the active site of the polymerase prevents thechemical incorporation of the nucleotide into the primer of the primedtemplate nucleic acid.

The examination step may be controlled, in part, by providing reactionconditions to prevent chemical incorporation of a nucleotide whileallowing monitoring of the interaction between the crippled DNApolymerase and the primed template nucleic acid, thereby permittingdetermination of the identity of the next base of the nucleic acidtemplate strand. Such reaction conditions may be referred to as“examination reaction conditions.” Optionally, a ternary complex orclosed-complex is formed under examination conditions. Optionally, astabilized ternary complex or closed-complex is formed under examinationconditions or in a pre-chemistry conformation. Optionally, a stabilizedclosed-complex is in a pre-translocation conformation, wherein theenclosed nucleotide has been incorporated, but the closed-complex doesnot allow for the incorporation of a subsequent nucleotide. Optionally,the examination conditions accentuate the difference in affinity forpolymerase to primed template nucleic acids in the presence of differentnucleotides. Optionally, the examination conditions cause differentialaffinity of the crippled DNA polymerase for the primed template nucleicacid in the presence of different nucleotides. By way of example, theexamination conditions that cause differential affinity of the crippledDNA polymerase for the primed template nucleic acid in the presence ofdifferent nucleotides include, but are not limited to, high salt andinclusion of potassium glutamate. Concentrations of potassium glutamatethat can be used to alter polymerase affinity for the primed templatenucleic acid include 10 mM to 1.6 M of potassium glutamate, or anyamount in between 10 mM and 1.6 M. Optionally, high salt refers to aconcentration of salt from 50 mM to 1,500 mM salt.

Examination typically involves, in the monitoring step, detectingcrippled DNA polymerase interaction with a template nucleic acid, orwith template nucleic acid and nucleotide in combination. Detection mayinclude optical, electrical, thermal, acoustic, chemical and mechanicalmeans. Optionally, monitoring is performed after a buffer change or awash step, wherein the wash step removes any non-bound reagents (e.g.,unbound polymerases and/or nucleotides) from the region of observation.Optionally, monitoring is performed during a buffer change or a washstep, such that the dissociation kinetics of the polymerase-nucleic acidor polymerase-nucleic acid-nucleotide complexes may be used to determinethe identity of the next base. Optionally, monitoring is performedduring the course of addition of the examination reaction mixture orfirst reaction mixture, such that the association kinetics of thepolymerase to the nucleic acid may be used to determine the identity ofthe next base on the nucleic acid. Optionally, monitoring involvesdistinguishing closed-complexes from binary complexes of polymerase andprimed template nucleic acid. Optionally, monitoring is performed underequilibrium conditions where the affinities measured are equilibriumaffinities. Multiple examination steps including different or similarexamination reagents, may be performed sequentially to ascertain theidentity of the next template base. Multiple examination steps may beutilized in cases where multiple template nucleic acids are beingsequenced simultaneously in one sequencing reaction, wherein differentnucleic acids react differently to the different examination reagents.Optionally, multiple examination steps may improve the accuracy of nextbase determination.

In an exemplary sequencing reaction, the examination step includesformation and/or stabilization of a closed-complex including a crippledDNA polymerase, a primed template nucleic acid, and the next correctnucleotide. Characteristics of the formation and/or release of theclosed-complex are monitored to identify the enclosed nucleotide andtherefore the next base in the template nucleic acid. Closed-complexcharacteristics can be dependent on the sequencing reaction components(e.g., crippled DNA polymerase, primer, template nucleic acid,nucleotide) and/or reaction mixture components and/or conditions.Optionally, the closed-complex is in a pre-chemistry conformation.Optionally, the closed-complex is in a pre-translocation conformation.Optionally, the closed-complex is in a post-translocation conformation.

The examination step involves monitoring the interaction of a crippledDNA polymerase with a primed template nucleic acid in the presence of atest nucleotide. The formation of a closed-complex may be monitored.Optionally, the absence of formation of a closed-complex is monitored.Optionally, the dissociation of a closed-complex is monitored.Optionally, the incorporation step involves monitoring incorporation ofa nucleotide. Optionally, the incorporation step involves monitoring theabsence of nucleotide incorporation.

Any process of the examination and/or incorporation step may bemonitored. Optionally, a crippled DNA polymerase has an exogenous labelor “tag.” Optionally, the detectable tag or label on the crippled DNApolymerase is removable. Optionally, the nucleotides or crippled DNApolymerases have a detectable label, however, the label is not detectedduring sequencing. Optionally, no component of the sequencing reactionis detectably labeled with an exogenous label.

Monitoring the variation in affinity of a crippled DNA polymerase for atemplate nucleic acid in the presence of correct and incorrectnucleotides, under conditions that do not allow the incorporation of thenucleotide, may be used to determine the sequence of the nucleic acid.The affinity of a crippled DNA polymerase for a template nucleic acid inthe presence of different nucleotides, including modified or labelednucleotides, can be monitored as the off-rate of the polymerase-nucleicacid interaction in the presence of the various nucleotides. Theaffinities and off-rates of many standard polymerases to variousmatched/correct, mismatched/incorrect and modified nucleotides are knownin the art. Single molecule imaging of Klenow polymerase reveals thatthe off-rate for a template nucleic acid for different nucleotide types,where the nucleotide types are prevented from incorporating, aredistinctly and measurably different.

Optionally, a nucleotide of a particular type is made available to acrippled DNA polymerase in the presence of a primed template nucleicacid. The reaction is monitored, wherein, if the nucleotide is a nextcorrect nucleotide, the polymerase may be stabilized to form aclosed-complex. If the nucleotide is an incorrect nucleotide, aclosed-complex may still be formed; however, without the additionalassistance of stabilizing agents or reaction conditions (e.g., absenceof catalytic ions, polymerase inhibitors, salt), the closed-complex maydissociate. The rate of dissociation is dependent on the affinity of theparticular combination of polymerase, template nucleic acid, andnucleotide, as well as reaction conditions. Optionally, the affinity ismeasured as an off-rate. Optionally, the affinity is different betweendifferent nucleotides for the closed-complex. For example, if the nextbase in the template nucleic acid downstream of the 3′-end of the primeris G, the polymerase-nucleic acid affinity, measured as an off-rate, isexpected to be different based on whether dATP, dCTP, dGTP or dTTP areadded. In this case, dCTP would have the slowest off-rate, with theother nucleotides providing different off-rates for the interaction.Optionally, the off-rate may be different depending on the reactionconditions, for example, the presence of stabilizing agents (e.g.,inhibitory compounds) or reaction conditions (e.g., nucleotidemodifications or modified polymerases). Once the identity of the nextcorrect nucleotide is determined, 1, 2, 3, 4 or more nucleotide typesmay be introduced simultaneously to the reaction mixture underconditions that specifically target the formation of a closed-complex.Excess nucleotides may be removed from the reaction mixture and thereaction conditions modulated (e.g., by use of reversible terminatornucleotides) to incorporate the next correct nucleotide of theclosed-complex. This sequencing reaction ensures that only onenucleotide is incorporated per sequencing cycle.

The affinity of a crippled DNA polymerase for a template nucleic acid inthe presence of a nucleotide can be measured in a plurality of methodsknown to one of skill in the art. Optionally, the affinity is measuredas an off-rate, where the off-rate is measured by monitoring the releaseof the crippled DNA polymerase from the template nucleic acid as thereaction is washed by a wash buffer. The polymerase binding rates may bediffusion limited at sufficiently low concentrations of crippled DNApolymerase, wherein if the crippled DNA polymerase falls off from theDNA-polymerase complex, it does not load back immediately, allowing forsufficient time to detect that the polymerase has been released from thecomplex. For a higher affinity interaction, the crippled DNA polymeraseis released from the nucleic acid slowly, whereas a low affinityinteraction results in the polymerase being released more rapidly. Thespectrum of affinities, in this case, translates to different off-rates,with the off-rates measured under dynamic wash conditions or atequilibrium. The smallest off-rate corresponds to the base complementaryto the added nucleotide, while the other off-rates vary, in a knownfashion, depending on the combination of crippled DNA polymerase andnucleotide selected.

Optionally, the off-rate is measured as an equilibrium signal intensityafter the crippled DNA polymerase and nucleotide are provided in thereaction mixture, wherein the interaction with the lowest off-rate(highest affinity) nucleotide produces the strongest signal, while theinteractions with other, varying, off-rate nucleotides produce signalsof measurably different intensities. As a non-limiting example, afluorescently labeled polymerase, measured, preferably, under totalinternal reflection (TIRF) conditions, produces different measuredfluorescence intensities depending on the number of polymerase moleculesbound to surface-immobilized nucleic acid molecules in a suitably chosenwindow of time. The intrinsic fluorescence of the polymerase, forinstance, tryptophan fluorescence, may also be utilized. A high off-rateinteraction produces low measured intensities, as the number of boundcrippled DNA polymerase molecules, in the chosen time window is verysmall, wherein a high off-rate indicates that most of the polymerase isunbound from the nucleic acid. Any surface localized measurement schememay be employed including, but not limited to, labeled or fluorescenceschemes. Suitable measurement schemes that measure affinities underequilibrium conditions include, but are not limited to, bound mass,refractive index, surface charge, dielectric constant, and other schemesknown in the art. Optionally, a combination of on-rate and off-rateengineering yields higher fidelity detection in the proposed schemes. Asa non-limiting example, a uniformly low on-rate, base-dependent, varyingoff-rate results in an unbound polymerase remaining unbound forprolonged periods, allowing enhanced discrimination of the variation inoff-rate and measured intensity. The on-rate may be manipulated bylowering the concentration of the added crippled DNA polymerase,nucleotide, or both polymerase and nucleotide.

Optionally, the interaction between the crippled DNA polymerase and thenucleic acid is monitored via a detectable tag attached to thepolymerase. The tag may be monitored by detection methods including, butlimited to, optical, electrical, thermal, mass, size, charge, vibration,and pressure. The label may be magnetic, fluorescent or charged. Forexternal and internal label schemes, fluorescence anisotropy may be usedto determine the stable binding of a crippled DNA polymerase to anucleic acid in a closed-complex.

By way of example, a crippled DNA polymerase is tagged with afluorophore, wherein closed-complex formation is monitored as a stablefluorescent signal. The unstable interaction of the crippled DNApolymerase with the template nucleic acid in the presence of anincorrect nucleotide results in a measurably weaker signal compared tothe closed-complex formed in the presence of the next correctnucleotide. In certain preferred embodiments, however, thesequencing-by-binding procedure does not rely on detection of anyexogenous label (e.g., a fluorescent label) joined to the crippled DNApolymerase.

Optionally, a primed template nucleic acid molecule (optionally blockedat its 3′-end) is contacted with a crippled DNA polymerase and one ormore exogenously labeled nucleotides during the examination step.Monitoring of signal generated as a consequence of the presence of thelabeled nucleotide provides information concerning formation andstabilization/destabilization of the ternary complex that includes thelabeled nucleotide. For example, if the exogenous label is a fluorescentlabel, and if the primed template nucleic acid is immobilized to a solidsupport at a particular locus, then monitoring fluorescent signalassociated with that locus can be used for monitoring ternary complexformation and stability under different reaction mixture conditions.

The Identifying Step

The identity of the next correct base or nucleotide can be determined bymonitoring the presence, formation and/or dissociation of the ternarycomplex or closed-complex. The identity of the next base may bedetermined without chemically incorporating the next correct nucleotideinto the 3′-end of the primer. Optionally, the identity of the next baseis determined by monitoring the affinity of the crippled DNA polymerasefor the primed template nucleic acid in the presence of addednucleotides. Optionally, the affinity of the polymerase for the primedtemplate nucleic acid in the presence of the next correct nucleotide maybe used to determine the next correct base on the template nucleic acid.Optionally, the affinity of the crippled DNA polymerase for the primedtemplate nucleic acid in the presence of an incorrect nucleotide may beused to determine the next correct base on the template nucleic acid.

In certain embodiments, a ternary complex that includes a primedtemplate nucleic acid (or a blocked primed template nucleic acid) isformed in the presence of a crippled DNA polymerase and a plurality ofnucleotides. Cognate nucleotide participating in the ternary complexoptionally is identified by observing loss of the complex that occurswhen the cognate nucleotide is withdrawn from the reaction mixture, forexample by exchanging one reaction mixture for another. Here,destabilization of the complex is an indicator of cognate nucleotideidentity. Loss of binding signal (e.g., a fluorescent binding signalassociated with a particular locus on a solid support) can occur whenthe primed template nucleic acid is exposed to a reaction mixture thatdoes not include the cognate nucleotide. Optionally, maintenance of aternary complex in the presence of a single nucleotide in a reactionmixture also can indicate identity of the cognate nucleotide.

The Incorporation Step

Optionally, the methods provided herein further include an incorporationstep. By way of example, the incorporation step includes incorporating asingle nucleotide (e.g., an unlabeled nucleotide, a reversibleterminator nucleotide, or a detectably labeled nucleotide analog)complementary to the next base of the template nucleic acid into theprimer of the primed template nucleic acid molecule. Optionally, theincorporation step includes contacting the primed template nucleic acidmolecule, polymerase (other than the crippled DNA polymerase used in theexamination step) and nucleotide with an incorporation reaction mixture.The incorporation reaction mixture, typically includes a catalytic metalion.

The provided method may further include preparing the primed templatenucleic acid molecule for a next examination step after theincorporation step. Optionally, the preparing includes subjecting theprimed template nucleic acid or the nucleic acid/polymerase complex toone or more wash steps; a temperature change; a mechanical vibration; apH change; salt or buffer composition changes, an optical stimulation ora combination thereof. Optionally, the wash step includes contacting theprimed template nucleic acid or the primed template nucleicacid/polymerase complex with one or more buffers, detergents, proteindenaturants, proteases, oxidizing agents, reducing agents, or otheragents capable of releasing internal crosslinks within a polymerase orcrosslinks between a polymerase and nucleic acid.

Optionally, the method further includes repeating the examination stepand the incorporation step to sequence a template nucleic acid molecule.The examination step may be repeated one or more times prior toperforming the incorporation step. Optionally, two consecutiveexamination steps include reaction mixtures with different nucleotidemolecules (e.g., different nucleotides that are labeled or unlabeled).Optionally, prior to incorporating the single nucleotide into the primedtemplate nucleic acid molecule, the first reaction mixture is replacedwith a second reaction mixture including a polymerase capable ofphosphodiester bond formation and 1, 2, 3, or 4 types of nucleotidemolecules (e.g., different unlabeled nucleotides). Optionally, thenucleotide molecules are native nucleotides selected from dATP, dTTP,dCTP, and dGTP.

The incorporation reaction may be enabled by an incorporation reactionmixture. Optionally, the incorporation reaction mixture includes adifferent composition of nucleotides than the examination reaction. Forexample, the examination reaction includes one type of nucleotide andthe incorporation reaction includes another type of nucleotide. By wayof another example, the examination reaction includes one type ofnucleotide and the incorporation reaction includes four types ofnucleotides, or vice versa. Optionally, the examination reaction mixtureis altered or replaced by the incorporation reaction mixture.Optionally, the incorporation reaction mixture includes a catalyticmetal ion, potassium chloride, or a combination thereof.

Optionally, the incorporation step includes replacing a nucleotide fromthe examination step and incorporating another nucleotide into the3′-end of the template nucleic acid primer. The incorporation step canfurther involve releasing a nucleotide from within a closed-complex(e.g., the nucleotide is a modified nucleotide or nucleotide analog) andincorporating a nucleotide of a different kind to the 3′-end of thetemplate nucleic acid primer. Optionally, the released nucleotide isremoved and replaced with an incorporation reaction mixture including anext correct nucleotide.

Suitable reaction conditions for incorporation may involve replacing theexamination reaction mixture with an incorporation reaction mixture.Optionally, nucleotides present in the examination reaction mixture arereplaced with one or more nucleotides in the incorporation reactionmixture. Optionally, the polymerase present during the examination stepis replaced during the incorporation step. Optionally, the polymerasepresent during the examination step is modified during the incorporationstep. Optionally, the one or more nucleotides present during theexamination step are modified during the incorporation step. Thereaction mixture and/or reaction conditions present during theexamination step may be altered by any means during the incorporationstep. These means include, but are not limited to, removing reagents,chelating reagents, diluting reagents, adding reagents, alteringreaction conditions such as conductivity or pH, and any combinationthereof. The reagents in the reaction mixture including any combinationof polymerase, primed template nucleic acid, and nucleotide may bemodified during the examination step and/or incorporation step.

Optionally, the reaction mixture of the incorporation step includescompetitive inhibitors, wherein the competitive inhibitors reduce theoccurrence of multiple incorporations. In certain embodiments, thecompetitive inhibitor is a non-incorporable nucleotide. In certainembodiments, the competitive inhibitor is an aminoglycoside. Thecompetitive inhibitor is capable of replacing either the nucleotide orthe catalytic metal ion in the active site, such that after the firstincorporation the competitive inhibitor occupies the active sitepreventing a second incorporation. In some embodiments, both anincorporable nucleotide and a competitive inhibitor are introduced inthe incorporation step, such that the ratio of the incorporablenucleotide and the inhibitor can be adjusted to ensure incorporation ofa single nucleotide at the 3′-end of the primer.

Optionally, the provided reaction mixtures, including the incorporationreaction mixtures, include at least one unlabeled nucleotide moleculethat is a non-incorporable nucleotide. In other words, the providedreaction mixtures can include one or more unlabeled nucleotide moleculesthat are incapable of incorporation into the primer of the primedtemplate nucleic acid molecule. Nucleotides incapable of incorporationinclude, for example, diphosphate nucleotides. For instance, thenucleotide may contain modifications to the triphosphate group that makethe nucleotide non-incorporable. Examples of non-incorporablenucleotides may be found in U.S. Pat. No. 7,482,120, the disclosure ofwhich is incorporated by reference herein in its entirety. Optionally,the primer may not contain a free hydroxyl group at its 3′-end, therebyrendering the primer incapable of incorporating any nucleotide, and,thus making any nucleotide non-incorporable.

A polymerase inhibitor optionally may be included with the reactionmixtures containing test nucleotides in the examination step to trap thepolymerase on the nucleic acid upon binding the next correct nucleotide.Optionally, the polymerase inhibitor is a pyrophosphate analog.Optionally, the polymerase inhibitor is an allosteric inhibitor.Optionally, the polymerase inhibitor is a DNA or an RNA aptamer.Optionally, the polymerase inhibitor competes with a catalytic-ionbinding site in the polymerase. Optionally, the polymerase inhibitor isa reverse transcriptase inhibitor. The polymerase inhibitor may be anHIV-1 reverse transcriptase inhibitor or an HIV-2 reverse transcriptaseinhibitor. The HIV-1 reverse transcriptase inhibitor may be a(4/6-halogen/MeO/EtO-substituted benzo[d]thiazol-2-yl)thiazolidin-4-one.

In the provided sequencing methods, the next correct nucleotide isidentified before the incorporation step, allowing the incorporationstep to not require labeled reagents and/or monitoring. Thus, in theprovided methods, a nucleotide, optionally, does not contain an attacheddetectable tag or label. Optionally, the nucleotide contains adetectable label, but the label is not detected in the method.Optionally, the correct nucleotide does not contain a detectable label;however, an incorrect or non-complementary nucleotide to the next basecontains a detectable label.

The examination step of the sequencing reaction may be repeated 1, 2, 3,4 or more times prior to the incorporation step. The examination andincorporation steps may be repeated until the desired sequence of thetemplate nucleic acid is obtained.

Reaction Mixtures

Nucleic acid sequencing reaction mixtures, or simply “reactionmixtures,” typically include reagents that are commonly present inpolymerase-based nucleic acid synthesis reactions. Reaction mixturereagents include, but are not limited to, enzymes (e.g., the crippledDNA polymerase, or the polymerase used in the incorporation step),dNTPs, template nucleic acids, primer nucleic acids, salts, buffers,small molecules, co-factors, metals, and ions. The ions may be catalyticions, divalent catalytic ions, non-catalytic ions, non-covalent metalions, or a combination thereof. The reaction mixture can include saltssuch as NaCl, KCl, potassium acetate, ammonium acetate, potassiumglutamate, NH₄Cl, or NH₄HSO₄. The reaction mixture can include a sourceof ions, such as Mg²⁺ or Mn²⁺ Mg-acetate, Co²⁺ or Ba²⁺. The reactionmixture can include tin ions, Ca²⁺, Zn²⁺, Cu²⁺, Co²⁺, Fe²⁺, Ni²⁺, orEu⁺³. The buffer can include Tris, Tricine, HEPES, MOPS, ACES, MES,phosphate-based buffers, and acetate-based buffers. The reaction mixturecan include chelating agents such as EDTA, EGTA, and the like.Optionally, the reaction mixture includes cross-linking reagents.Provided herein are reaction mixtures, optionally, used during theexamination step, as well as incorporation reaction mixtures used duringnucleotide incorporation that can include one or more of theaforementioned agents. Reaction mixtures, when used during examination,can be referred to herein as examination reaction mixtures. Optionally,the examination reaction mixture includes a high concentration of salt;a high pH; 1, 2, 3, 4, or more types of unlabeled nucleotides; potassiumglutamate; a chelating agent; a polymerase inhibitor; a catalytic metalion; a non-catalytic metal ion that inhibits polymerase-mediatedincorporation; or any combination thereof. The examination reactionmixture can include 10 mM to 1.6 M of potassium glutamate or any amountin between 10 mM and 1.6 M. Optionally, the incorporation reactionmixture includes a catalytic metal ion; 1, 2, 3, 4, or more types ofnucleotides (e.g., unlabeled nucleotides); potassium chloride; anon-catalytic metal ion that inhibits polymerase-mediated incorporation;or any combination thereof.

Optionally, reaction mixtures in accordance with the disclosedtechniques modulate the formation and stabilization of a closed-complexduring an examination step. For example, the reaction conditions of theexamination step optionally can favor the formation and/or stabilizationof a closed-complex encapsulating a nucleotide, and hinder the formationand/or stabilization of a binary complex. The binary interaction betweenthe polymerase and template nucleic acid may be manipulated bymodulating sequencing reaction parameters such as ionic strength, pH,temperature, or any combination thereof, or by the addition of a binarycomplex destabilizing agent to the reaction. Optionally, high salt(e.g., 50 mM to 1,500 mM) and/or pH changes are utilized to destabilizea binary complex. Optionally, a binary complex may form between apolymerase and a template nucleic acid during the examination orincorporation step of the sequencing reaction, regardless of thepresence of a nucleotide. Optionally, the reaction conditions favor thestabilization of a closed ternary complex and destabilization of abinary complex. By way of example, the pH of the examination reactionmixture can be adjusted from pH 4.0 to pH 10.0 to favor thestabilization of a closed ternary complex and destabilization of abinary complex. Optionally, the pH of the examination reaction mixtureis from pH 4.0 to pH 6.0. Optionally, the pH of the examination reactionmixture is pH 6.0 to pH 10.0.

The provided reaction mixtures and sequencing methods disclosed hereinencourage polymerase interaction with the nucleotides and templatenucleic acid in a manner that reveals the identity of the next basewhile controlling the chemical addition of a nucleotide. Optionally, themethods are performed in the absence of detectably labeled nucleotidesor in the presence of labeled nucleotides wherein the labels are notdetected. Optionally, the reaction mixtures include nucleotides thatharbor an exogenous detectable label (e.g., a fluorescent label).Optionally, a plurality of nucleotides in a reaction mixture harbor thesame exogenous detectable label. Optionally, a plurality of nucleotidesin a reaction mixture harbor different exogenous detectable labels.Optionally, the reaction mixtures can include one or more exogenouslylabeled polymerase enzymes.

Provided herein are reaction mixtures and methods that facilitateformation and/or stabilization of a closed-complex that includes apolymerase bound to a primed template nucleic acid and a nucleotideenclosed within the polymerase-template nucleic acid complex, underexamination reaction mixture conditions. Examination reaction conditionsmay inhibit or attenuate nucleotide incorporation. Optionally,incorporation of the enclosed nucleotide is inhibited and the complex isstabilized or trapped in a pre-chemistry conformation or a ternarycomplex. Optionally, the enclosed nucleotide is incorporated andsubsequent nucleotide incorporation is inhibited. In this instance, thecomplex is stabilized or trapped in a pre-translocation conformation.For the sequencing reactions provided herein, the closed-complex isstabilized during the examination step, allowing for controllednucleotide incorporation. Optionally, a stabilized closed-complex is acomplex wherein incorporation of an enclosed nucleotide is attenuated,either transiently (e.g., to examine the complex and then incorporatethe nucleotide) or permanently (e.g., for examination only) during anexamination step. Optionally, a stabilized closed-complex allows for theincorporation of the enclosed nucleotide, but does not allow for theincorporation of a subsequent nucleotide. Optionally, the closed-complexis stabilized in order to monitor any polymerase interaction with atemplate nucleic acid in the presence of a nucleotide for identificationof the next base in the template nucleic acid.

Optionally, the enclosed nucleotide has severely reduced or disabledbinding to the template nucleic acid in the closed-complex. Optionally,the enclosed nucleotide is base-paired to the template nucleic acid at anext base. Optionally, the identity of the polymerase, nucleotide,primer, template nucleic acid, or any combination thereof, affects theinteraction between the enclosed nucleotide and the template nucleicacid in the closed-complex.

Optionally, the enclosed nucleotide is bound to the polymerase of theclosed-complex. Optionally, the enclosed nucleotide is weakly associatedwith the polymerase of the closed-complex. Optionally, the identity ofthe polymerase, nucleotide, primer, template nucleic acid, or anycombination thereof, affects the interaction between the enclosednucleotide and the polymerase in the closed-complex. For a givenpolymerase, each nucleotide has a different affinity for the polymerasethan another nucleotide. Optionally, this affinity is dependent, inpart, on the template nucleic acid and/or the primer.

The closed-complex may be transiently formed. Optionally, the enclosednucleotide is a next correct nucleotide. In some methods, the presenceof the next correct nucleotide contributes, in part, to thestabilization of a closed-complex. Optionally, the enclosed nucleotideis not a next correct nucleotide.

Optionally, the examination reaction condition comprises a plurality ofprimed template nucleic acids, polymerases, nucleotides, or anycombination thereof. Optionally, the plurality of nucleotides comprises1, 2, 3, 4, or more types of different nucleotides, for example dATP,dTTP, dGTP, and dCTP. Optionally, the plurality of template nucleicacids is a clonal population of template nucleic acids.

Reaction conditions that may modulate the stability of a closed-complexinclude, but are not limited to, the availability of catalytic metalions, suboptimal or inhibitory metal ions, ionic strength, pH,temperature, polymerase inhibitors, cross-linking reagents, and anycombination thereof. Reaction reagents which may modulate the stabilityof a closed-complex include, but are not limited to, non-incorporablenucleotides, incorrect nucleotides, nucleotide analogs, modifiedpolymerases, template nucleic acids with non-extendible polymerizationinitiation sites, and any combination thereof.

The examination reaction mixture can include other molecules including,but not limited to, enzymes. Optionally, the examination reactionmixture includes any reagents or biomolecules generally present in anucleic acid polymerization reaction. Reaction components may include,but are not limited to, salts, buffers, small molecules, metals, andions. Optionally, properties of the reaction mixture may be manipulated,for example, electrically, magnetically, and/or with vibration.

Nucleotides and Nucleotide Analogs

Nucleotides useful for carrying out the sequencing-by-binding proceduresdescribed herein include native nucleotides, labeled nucleotides (e.g.,nucleotides that include an exogenous fluorescent dye or other label notfound in native nucleotides), and nucleotide analogs (e.g., nucleotideshaving a reversible terminator moiety).

There is flexibility in the nature of the nucleotides that may beemployed in connection with the presently described technique. Anucleotide may include as its nitrogenous base any of: adenine,cytosine, guanine, thymine, or uracil. Optionally, a nucleotide includesinosine, xanthanine, hypoxanthanine, isocytosine, isoguanine,nitropyrrole (including 3-nitropyrrole) or nitroindole (including5-nitroindole) base. Useful nucleotides include, but are not limited to,ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, GMP, dATP, dTTP,dCTP, dGTP, dUTP, dADP, dTDP, dCDP, dGDP, dUDP, dAMP, dTMP, dCMP, dGMP,and dUMP. Optionally, the phosphate group is modified with a moiety. Themoiety may include a detectable label. Optionally, the 3′ OH group ofthe nucleotide is modified with a moiety, where the moiety may be a 3′reversible or irreversible terminator moiety. Optionally, the 2′position of the nucleotide is modified with a moiety, where the moietymay be a 2′ reversible or irreversible terminator moiety. Optionally,the base of the nucleotide is modified to include a reversibleterminator moiety. Nucleotides may also contain terminating inhibitorsof DNA polymerase, dideoxynucleotides or 2′,3′ dideoxynucleotides, whichare abbreviated as ddNTPs (ddGTP, ddATP, ddTTP, ddCTP, and ddUTP).

Optionally, a closed-complex of an examination step includes anucleotide analog or modified nucleotide to facilitate stabilization ofthe closed-complex. Optionally, a nucleotide analog includes anitrogenous base, five-carbon sugar, and phosphate group and anycomponent of the nucleotide may be modified and/or replaced. Nucleotideanalogs may be non-incorporable nucleotides. Non-incorporablenucleotides may be modified to become incorporable at any point duringthe sequencing method.

Nucleotide analogs include, but are not limited to, alpha-phosphatemodified nucleotides, alpha-beta nucleotide analogs, beta-phosphatemodified nucleotides, beta-gamma nucleotide analogs, gamma-phosphatemodified nucleotides, caged nucleotides, or ddNTPs. Examples ofnucleotide analogs are described in U.S. Pat. No. 8,071,755, which isincorporated by reference herein in its entirety.

Nucleotide analogs can include terminators that reversibly preventnucleotide incorporation to the 3′-end of the primer. One type ofreversible terminator is a 3′-O-blocked reversible terminator. Theterminator is linked to the oxygen atom of the 3′ OH end of the 5-carbonsugar of a nucleotide. Another type of reversible terminator is a3′-unblocked reversible terminator. The terminator is linked to thenitrogenous base of a nucleotide. For reviews of nucleotide analogshaving terminators, see, e.g., Mu, R., et al., “The History and Advancesof Reversible Terminators Used in New Generations of SequencingTechnology,” Genomics, Proteomics & Bioinformatics 11(1):34-40 (2013),which is incorporated by reference herein in its entirety.

Optionally, nucleotides are substituted for modified nucleotide analogshaving terminators that irreversibly prevent nucleotide incorporation tothe 3′-end of the primer. Irreversible nucleotide analogs includedideoxynucleotides, ddNTPs (ddGTP, ddATP, ddTTP, ddCTP).Dideoxynucleotides lack the 3′-OH group of dNTPs that is essential forpolymerase-mediated synthesis.

Optionally, non-incorporable nucleotides include a blocking moiety thatinhibits or prevents the nucleotide from forming a covalent linkage to asecond nucleotide (3′ OH of a primer) during the incorporation step of anucleic acid polymerization reaction. The blocking moiety can be removedfrom the nucleotide, allowing for nucleotide incorporation.

Optionally, a nucleotide analog present in a closed-complex renders theclosed-complex stable. Optionally, the nucleotide analog isnon-incorporable. Optionally, the nucleotide analog is released and anative nucleotide is incorporated. Optionally, the closed-complex isreleased, the nucleotide analog is modified, and the modified nucleotideanalog is incorporated. Optionally, the closed-complex is released underreaction conditions that modify and/or destabilize the nucleotide analogin the closed-complex.

Optionally, a nucleotide analog present in a closed-complex isincorporated and the closed-complex is stabilized. The closed-complexmay be stabilized by the nucleotide analog, or for example, by anystabilizing methods disclosed herein. Optionally, the nucleotide analogdoes not allow for the incorporation of a subsequent nucleotide. Theclosed-complex can be released, for example, by any methods describedherein, and the nucleotide analog is modified. The modified nucleotideanalog may allow for subsequent incorporation of a nucleotide to its3′-end.

Optionally, a nucleotide analog is present in the reaction mixtureduring the examination step. For example, 1, 2, 3, 4 or more nucleotideanalogs are present in the reaction mixture during the examination step.Optionally, a nucleotide analog is replaced, diluted, or sequesteredduring an incorporation step. Optionally, a nucleotide analog isreplaced with a native nucleotide. The native nucleotide may include anext correct nucleotide. Optionally, a nucleotide analog is modifiedduring an incorporation step. The modified nucleotide analog can besimilar to or the same as a native nucleotide.

Optionally, a nucleotide analog has a different binding affinity for acrippled DNA polymerase than a native nucleotide. Optionally, anucleotide analog has a different interaction with a next base than anative nucleotide. Nucleotide analogs and/or non-incorporablenucleotides may base-pair with a complementary base of a templatenucleic acid.

Optionally, a nucleotide analog is a nucleotide, modified or native,fused to a polymerase. Optionally, a plurality of nucleotide analogsincludes fusions to a plurality of polymerases, wherein each nucleotideanalog includes a different polymerase.

A nucleotide can be modified to favor the formation of a closed-complexover the formation of a binary complex. A nucleotide may be selected ormodified to have a high affinity for a crippled DNA polymerase, whereinthe polymerase binds to a nucleotide prior to binding to the templatenucleic acid.

Any nucleotide modification that traps the crippled DNA polymerase in aclosed-complex may be used in the methods disclosed herein. Thenucleotide may be trapped permanently or transiently. Optionally, thenucleotide analog is not the means by which a closed-complex isstabilized. Any closed-complex stabilization method may be combined in areaction utilizing a nucleotide analog.

Optionally, a nucleotide analog that allows for the stabilization of aclosed-complex is combined with reaction conditions that usually releasethe closed-complex. The conditions include, but are not limited to, thepresence of a release reagent (e.g., catalytic metal ion, such asmagnesium or manganese). Optionally, the closed-complex is stabilizedeven in the presence of a catalytic metal ion. Optionally, theclosed-complex is released even in the presence of a nucleotide analog.Optionally, the stabilization of the closed-complex is dependent, inpart, on the concentrations and/or identity of the stabilization reagentand/or release reagents, and any combination thereof. Optionally, thestabilization of a closed-complex using nucleotide analogs is combinedwith additional reaction conditions that function to stabilize aclosed-complex, including, but not limited to, sequestering, removing,reducing, omitting, and/or chelating a catalytic metal ion; the presenceof a polymerase inhibitor, cross-linking agent; and any combinationthereof.

Optionally, one or more nucleotides can be labeled with distinguishingand/or detectable tags or labels; however, such tags or labels are notdetected during examination, identification of the base or incorporationof the base, and are not detected during the sequencing methodsdisclosed herein. The tags may be distinguishable by means of theirdifferences in fluorescence, Raman spectrum, charge, mass, refractiveindex, luminescence, length, or any other measurable property. The tagmay be attached to one or more different positions on the nucleotide, solong as the fidelity of binding to the polymerase-nucleic acid complexis sufficiently maintained to enable identification of the complementarybase on the template nucleic acid correctly. Optionally, the tag isattached to the nucleobase position of the nucleotide. Under suitablereaction conditions, the tagged nucleotides may be enclosed in aclosed-complex with the polymerase and the primed template nucleic acid.Alternatively, a tag is attached to the gamma phosphate position of thenucleotide.

Polymerases

Crippled DNA polymerases useful for carrying out the disclosedsequencing-by-binding technique include modified variants of naturallyoccurring polymerases, including, but not limited to, mutants,recombinants, fusions, genetic modifications, chemical modifications,synthetics, and analogs. Optionally, the modified variants have specialproperties that enhance their ability to sequence DNA, includingenhanced binding affinity to nucleic acids, reduced binding affinity tonucleic acids, enhanced catalysis rates, reduced catalysis rates etc.Mutant polymerases include polymerases wherein one or more amino acidsare replaced with other amino acids (naturally or non-naturallyoccurring), one or more amino acids are chemically modified, and/or oneor more amino acids are inserted or deleted. Modified polymerasesinclude polymerases that contain an external tag, which can be used tomonitor the presence and interactions of the polymerase. Optionally,intrinsic signals from the polymerase can be used to monitor theirpresence and interactions. Thus, the provided methods can includemonitoring the interaction of the polymerase, nucleotide and templatenucleic acid through detection of an intrinsic signal from thepolymerase. Optionally, the intrinsic signal is a light scatteringsignal. For example, intrinsic signals include native fluorescence ofcertain amino acids such as tryptophan, wherein changes in intrinsicsignals from the polymerase may indicate the formation of aclosed-complex. Thus, in the provided methods, the polymerase can be anunlabeled polymerase, and monitoring can be performed in the absence ofa detectable label associated with the polymerase.

The term polymerase and its variants, as used herein, also refers tofusion proteins including at least two portions linked to each other,for example, where one portion includes a polypeptide that can catalyzethe polymerization of nucleotides into a nucleic acid strand is linkedto another portion that includes a second moiety, such as, a reporterenzyme or a processivity-modifying domain. For example, T7 DNApolymerase includes a nucleic acid polymerizing domain and a thioredoxinbinding domain, wherein thioredoxin binding enhances the processivity ofthe polymerase. Absent the thioredoxin binding, T7 DNA polymerase is adistributive polymerase with processivity of only one to a few bases.Although DNA polymerases differ in detail, they have a similar overallshape of a hand with specific regions referred to as the fingers, thepalm, and the thumb; and a similar overall structural transition,including the movement of the thumb and/or finger domains, during thesynthesis of nucleic acids.

DNA polymerases that may be modified to yield crippled DNA polymerasesinclude, but are not limited to, bacterial DNA polymerases, eukaryoticDNA polymerases, archaeal DNA polymerases, viral DNA polymerases andphage DNA polymerases. Bacterial DNA polymerases include E. coli DNApolymerases I, II and III, IV and V, the Klenow fragment of E. coli DNApolymerase, Clostridium stercorarium (Cst) DNA polymerase, Clostridiumthermocellum (Cth) DNA polymerase and Sulfolobus solfataricus (Sso) DNApolymerase. Eukaryotic DNA polymerases include DNA polymerases α, β, γ,δ, ∈, η, ξ, λ, σ, μ, and k, as well as the Revl polymerase (terminaldeoxycytidyl transferase) and terminal deoxynucleotidyl transferase(TdT). Viral DNA polymerases include T4 DNA polymerase, phi-29 DNApolymerase, GA-1, phi-29-like DNA polymerases, PZA DNA polymerase,phi-15 DNA polymerase, Cpl DNA polymerase, Cpl DNA polymerase, T7 DNApolymerase, and T4 polymerase. Other DNA polymerases includethermostable and/or thermophilic DNA polymerases such as DNA polymerasesisolated from Thermus aquaticus (Taq) DNA polymerase, Thermus filiformis(Tfi) DNA polymerase, Thermococcus zilligi (Tzi) DNA polymerase, Thermusthermophilus (Tth) DNA polymerase, Thermus flavusu (Tfl) DNA polymerase,Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNApolymerase and Turbo Pfu DNA polymerase, Thermococcus litoralis (Tli)DNA polymerase, Pyrococcus sp. GB-D polymerase, Thermotoga maritima(Tma) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase,Pyrococcus Kodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase,Thermococcus sp. JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius(Tgo) DNA polymerase, Thermococcus acidophilium DNA polymerase;Sulfolobus acidocaldarius DNA polymerase; Thermococcus sp. go N-7 DNApolymerase; Pyrodictium occultum DNA polymerase; Methanococcus voltaeDNA polymerase; Methanococcus thermoautotrophicum DNA polymerase;Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNApolymerase (D. Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcushorikoshii DNA polymerase; Pyrococcus islandicum DNA polymerase;Thermococcus fumicolans DNA polymerase; Aeropyrum pernix DNA polymerase;and the heterodimeric DNA polymerase DP1/DP2. Engineered and modifiedpolymerases also are useful in connection with the disclosed techniques.For example, modified versions of the extremely thermophilic marinearchaea Thermococcus species 9° N (e.g., Therminator DNA polymerase fromNew England BioLabs Inc.; Ipswich, Mass.) can be used. Still otheruseful DNA polymerases, including the 3PDX polymerase are disclosed inU.S. Pat. No. 8,703,461, the disclosure of which is incorporated byreference in its entirety.

RNA polymerases include, but are not limited to, viral RNA polymerasessuch as T7 RNA polymerase, T3 polymerase, SP6 polymerase, and Kllpolymerase; Eukaryotic RNA polymerases such as RNA polymerase I, RNApolymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymeraseV; and Archaea RNA polymerases.

Reverse transcriptases include, but are not limited to, HIV-1 reversetranscriptase from human immunodeficiency virus type 1 (PDB 1HMV), HIV-2reverse transcriptase from human immunodeficiency virus type 2, M-MLVreverse transcriptase from the Moloney murine leukemia virus, AMVreverse transcriptase from the avian myeloblastosis virus, andtelomerase reverse transcriptase that maintains the telomeres ofeukaryotic chromosomes.

Modeling Catalytically Inactive Polymerase Mutants

Described below are the preparation of DNA polymerase I (pol I) largefragment mutants from a thermostable family strain of Bacillusstearothermophilus (Bst-f), where the mutants properly form ternarycomplexes with cognate nucleotides, but are incapable of incorporatingthose nucleotides in the presence of Mg²⁺ ions. The Bst-f enzyme is afamily A polymerase having homology to other well-characterized, highfidelity polymerases, including E. coli DNA pol I (KF), T. aquaticus DNApol I (Taq), and Bacillus subtilis DNA pol I (Bsu-f). These polymerasesshare conserved protein sequence motifs needed to fulfill distinctfunctions, including nucleotide binding and polymerization.

Further, the family A polymerases share common structural architecturesknown as the fingers, thumb and palm subdomains. The palm subdomain ishighly conserved among DNA polymerases, as it includes the catalyticcore that will be familiar to those having an ordinary level of skill inthe art. The palm also contains catalytic carboxylates from motifs A andC. The conserved acidic amino acid residues, aspartic acid (D) andglutamic acid (E), in motifs A and C are thought to serve as metalligands for divalent cations during phosphoryl transfer reactioncatalyzed by polymerases. Motif A contains a strictly-conservedaspartate at the junction of a beta-strand and an alpha-helix, and motifC has a doublet of negative charges, located in a beta-turn-betasecondary structure.

The parent enzyme (“CBT”) used for preparing mutants was an engineeredversion of the Bst polymerase that had been optimized with respect tocysteine content, and N-terminal sequences that facilitated proteinpurification and processing. More specifically, the polypeptide sequenceidentified as SEQ ID NO:1 included a modified N-terminus having: (1) anengineered “His-tag” motif at positions 5-10; (2) a thrombin cleavagesite between positions 17 and 18; and (3) a cysteine residue at position23. The naturally occurring Bst polymerase sequence extended fromposition 27 to the C-terminus (subject to removal of naturally occurringcysteine residues). It is to be understood that engineered polymerasesin accordance with the disclosure optionally include or omit theabove-described N-terminal modifications. For example, usefulpolymerases can be constructed on a parent scaffold of SEQ ID NO:2(omits sequences upstream of the thrombin cleavage site) or SEQ ID NO:3(omits sequences upstream of the first amino acid of the native Bstpolymerase). Thus, useful modifications that affect capacity forphosphodiester bond formation can be understood with reference to theseprotein sequence scaffolds.

Arrangements of the conventional A and C motifs are presented below.Both alignments were prepared using standard polypeptide sequencealignment software tools. The underlined and bolded aspartic acid (D)residue in motif A corresponds to amino acid position 653 in the crystalstructure of the Bst polymerase (UniProt No. P52026, Protein Data BankNo. 3EZ5). The underlined and bolded aspartic acid and glutamic acid(DE) residues in motif C correspond to amino acid positions 830-831 inthe crystal structure of the Bst polymerase (UniProt No. P52026, ProteinData Bank No. 3EZ5).

Motif A Klenow Fragment DYVIVSA D YSQIELRIMAHLSRDKGL (SEQ ID NO: 4) TaqGWLLVAL D YSQIELRVLAHLSGDENL (SEQ ID NO: 5) Bst-f DWLIFAA DYSQIELRVLAHIAEDDNL (SEQ ID NO: 6) Bsu-f DWLIFAA D YSQIELRVLAHISKDENL(SEQ ID NO: 7) Motif C Klenow Fragment MIMQVH DE LVFEVHKDDVD(SEQ ID NO: 8) Taq MLLQVH DE LVLEAPKERAE (SEQ ID NO: 9) Bst-f LLLQVH DELILEAPKEEIE (SEQ ID NO: 10) Bsu-f LLLQVH DE LIFEAPKEEIE (SEQ ID NO: 11)

The Bst-f numbering for the bolded and underlined motif A aspartate (D)residue is residue 381 in SEQ ID NO:1 (or residue 364 in SEQ ID NO:2; orresidue 355 in SEQ ID NO:3). The motif C aspartate (D) and glutamate (E)residues are numbered 558 and 559 in SEQ ID NO:1 (or residues 541 and542 in SEQ ID NO:2; or residues 532 and 533 in SEQ ID NO:3),respectively. While most DNA polymerases are known to include aminoacids providing three carboxylate side chains in motifs A and C, somerequire only two carboxylate side chains during the catalysis. Residues381 and 558 of SEQ ID NO:1 were picked for mutagenesis to investigatetheir effects on polymerase activity. These residues were substituted,using site-directed mutagenesis and an expression vector encoding thepolymerase protein, with either glutamate (E) or asparagine (N). Again,the objective was to allow maintenance of the DNA and dNTP bindingproperties while inhibiting the polymerization chemistry step. A summaryof key mutations is presented in Table 1.

TABLE 1 Summary of Key Mutations Mutant Name Mutation Position CBTCys-optimized Bst N/A enzyme with N- terminal modifications TDE D to Ein Motif A 381 of SEQ ID NO: 1 364 of SEQ ID NO: 2 355 of SEQ ID NO: 3BDE D to E in Motif C 558 of SEQ ID NO: 1 541 of SEQ ID NO: 2 532 of SEQID NO: 3 TDN D to N in Motif A 381 of SEQ ID NO: 1 364 of SEQ ID NO: 2355 of SEQ ID NO: 3

As described briefly below, the TDE and BDE mutant polymerases behavedsubstantially similarly. More specifically, both mutants were useful forbinding and identifying cognate nucleotide during an examinationreaction without incorporation. As well, neither mutant possessedcatalytic incorporation activity in the presence of Mg²⁺ ions. Incontrast, the TDN mutant was inactive (i.e., being incapable ofidentifying cognate nucleotide in an examination step).

Crippled DNA Polymerase Reporters

Optionally, a crippled DNA polymerase is tagged with a luminescent tag,wherein closed-complex formation is monitored as a stable luminescencesignal in the presence of the appropriate luminescence triggers (e.g., aradiation trigger or, in the case of chemiluminescent tags, chemicaltrigger). The unstable interaction of the crippled DNA polymerase withthe template nucleic acid in the presence of an incorrect nucleotideresults in a measurably weaker signal compared to the closed-complexformed in the presence of the next correct nucleotide. Additionally, awash step prior to triggering luminescence could remove all polymerasemolecules not bound in a stable closed-complex.

Optionally, a crippled DNA polymerase is tagged with an opticalscattering tag, wherein closed-complex formation is monitored as astable optical scattering signal. The unstable interaction of thecrippled DNA polymerase with the nucleic acid in the presence of anincorrect nucleotide results in a measurably weaker signal compared tothe closed-complex formed in the presence of the next correctnucleotide.

Optionally, the crippled DNA polymerase is tagged with a plasmonicnanoparticle tag, wherein the closed-complex formation is monitored as ashift in plasmonic resonance that is different from the plasmonicresonance in the absence of the closed-complex or the presence of aclosed-complex including an incorrect nucleotide. The change in plasmonresonance may be due to the change in local dielectric environment inthe closed-complex, or it may be due to the synchronous aggregation ofthe plasmonic nanoparticles on a cluster of clonally amplified nucleicacid molecules or another means that affects the plasmons differently inthe closed-complex configuration.

Optionally, the crippled DNA polymerase is tagged with a Ramanscattering tag, wherein the closed-complex formation is monitored as astable Raman scattering signal. The unstable interaction of crippled DNApolymerase with the nucleic acid in the presence of an incorrectnucleotide results in a measurably weaker signal compared to theclosed-complex formed in the presence of the next correct nucleotide.

Optionally, a next correct nucleotide is identified by a tag on acrippled DNA polymerase selected or modified to have a high affinity fornucleotides, wherein the polymerase binds to a nucleotide prior tobinding to the template nucleic acid. For example, the DNA polymerase Xfrom the African Swine Fever virus has an altered order of substratebinding, where the polymerase first binds to a nucleotide, then binds tothe template nucleic acid. Optionally, a polymerase is incubated witheach type of nucleotide in separate compartments, where each compartmentcontains a different type of nucleotide and where the polymerase islabeled differently with a tag depending on the nucleotide with which itis incubated. In these conditions, unlabeled nucleotides are bound todifferently labeled polymerases. The polymerases may be the same kind ofpolymerase bound to each nucleotide type or different polymerases boundto each nucleotide type. The differentially tagged polymerase-nucleotidecomplexes may be added simultaneously to any step of the sequencingreaction. Each polymerase-nucleotide complex binds to a template nucleicacid whose next base is complementary to the nucleotide in thepolymerase-nucleotide complex. The next correct nucleotide is identifiedby the tag on the polymerase carrying the nucleotide. The interrogationof the next template base by the labeled polymerase-nucleotide complexmay be performed under non-incorporating and/or examination conditions,where once the identity of the next template base is determined, thecomplex is destabilized and removed, sequestered, and/or diluted and aseparate incorporation step is performed in a manner ensuring that onlyone nucleotide is incorporated.

A common method of introducing a detectable tag on a polymeraseoptionally involves chemical conjugation to amines or cysteines presentin the non-active regions of the polymerase. Such conjugation methodsare well known in the art. As non-limiting examples,n-hydroxysuccinimide esters (NHS esters) are commonly employed to labelamine groups that may be found on an enzyme. Cysteines readily reactwith thiols or maleimide groups, while carboxyl groups may be reactedwith amines by activating them with EDC(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride).Optionally, N-hydroxysuccinimide (NHS) chemistry is employed at pHranges where only the N-terminal amines are reactive (for instance, pH7), such that only a single tag is added per polymerase.

Optionally, the tag attached to the crippled DNA polymerase is a chargetag, such that the formation of stable closed-complex can be detected byelectrical means by measuring changes in local charge density around thetemplate nucleic acids. Methods for detecting electrical charges arewell known in the art, including methods such as field-effecttransistors, dielectric spectroscopy, impedance measurements, and pHmeasurements, among others. Field-effect transistors include, but arenot limited to, ion-sensitive field-effect transistors (ISFET),charge-modulated field-effect transistors, insulated-gate field-effecttransistors, metal oxide semiconductor field-effect transistors andfield-effect transistors fabricated using semiconducting single wallcarbon nanotubes.

Optionally, a charge tag is a peptide tag having an isoelectric pointbelow about 4 or above about 10. Optionally, a crippled DNA polymeraseincluding a peptide tag has a total isoelectric point below about 5 orabove about 9. A charge tag may be any moiety which is positively ornegatively charged. The charge tag may include additional moietiesincluding mass and/or labels such as dyes. Optionally, the charge tagpossesses a positive or negative charge only under certain reactionconditions such as changes in pH.

A crippled DNA polymerase may be labeled with a fluorophore and/orquencher. Optionally, a nucleic acid is labeled with a fluorophoreand/or quencher. Optionally, one or more nucleotides are labeled with afluorophore and/or quencher. Exemplary fluorophores include, but are notlimited to, fluorescent nanocrystals; quantum dots; d-Rhodamine acceptordyes including dichloro[R110], dichloro[R6G], dichloro[TAMRA],dichloro[ROX] or the like; fluorescein donor dye including fluorescein,6-FAM, or the like; Cyanine dyes such as Cy3B; Alexa dyes, SETA dyes,Atto dyes such as atto 647N which forms a FRET pair with Cy3B and thelike. Fluorophores include, but are not limited to, MDCC(7-diethylamino-3-[([(2-maleimidyl)ethyl]amino)carbonyl]coumarin), TET,HEX, Cy3, TMR, ROX, Texas Red, Cy5, LC red 705 and LC red 640.Fluorophores and methods for their use including attachment topolymerases and other molecules are described in The Molecular Probes®Handbook (Life Technologies; Carlsbad Calif.) and Fluorophores Guide(Promega; Madison, Wis.), which are incorporated herein by reference intheir entireties. Exemplary quenches include, but are not limited to,ZEN, IBFQ, BHQ-1, BHQ-2, DDQ-I, DDQ-11, Dabcyl, Qx1 quencher, Iowa BlackRQ, and IRDye QC-1.

Optionally, a conformationally sensitive dye may be attached close tothe active site of the crippled DNA polymerase without affecting thepolymerization ability or fidelity of the polymerase; wherein a changein conformation, or a change in polar environment due to the formationof a closed-complex is reflected as a change in fluorescence orabsorbance properties of the dye. Common fluorophores such as Cy3 andfluorescein are known to have strong solvatochromatic response topolymerase binding and closed-complex formation, to the extent that theformation of closed-complex can be distinguished clearly from the binarypolymerase-nucleic acid complex. Optionally, the closed-complex can bedistinguished from binary complexes based on differences in fluorescenceor absorbance signals from a conformationally sensitive dye. Optionally,a solvatochromatic dye may be employed to monitor conformationaltransitions; wherein the change in local polar environment induced bythe conformational change can be used as the reporter signal.Solvatochromatic dyes include, but are not limited to, Reichart's dye,IR44, merocyanine dyes (e.g., merocyanine 540),4-[2-N-substituted-1,4-hydropyridin-4-ylidine)ethylidene]cyclohexa-2,5-dien-1-one,red pyrazolone dyes, azomethine dyes, indoaniline dyes, diazamerocyaninedyes, indigoid dyes, as exemplified by indigo, and others as well asmixtures thereof. Methods to introduce dyes or fluorophores to specificsites of a polymerase are well known in the art. As a non-limitingexample, a procedure for site specific labeling of a T7 DNA polymerasewith a dye is provided by Tsai et al., in “Site-Specific Labeling of T7DNA Polymerase with a Conformationally Sensitive Fluorophore and Its Usein Detecting Single-Nucleotide Polymorphisms,” Analytical Biochemistry384: 136-144 (2009), which is incorporated by reference herein in itsentirety.

Optionally, a polymerase is tagged with a fluorophore at a position thatcould sense closed-complex formation without interfering with thereaction. The polymerase may be a native or modified polymerase.Modified polymerases include those with one or more amino acid chemicalmodifications, mutations, additions, and/or deletions. Optionally, oneor more, but not all, cysteine amino acids are mutated to another aminoacid, such as alanine. In this case, the remaining one or more cysteinesare used for site-specific conjugation to a fluorophore. Alternatively,one or more amino acids are mutated to a reactive amino acid suitablefor fluorophore conjugation, such as cysteines or amino acids includingprimary amines.

Optionally, binding between a crippled DNA polymerase and a templatenucleic acid in the presence of a correct nucleotide may induce adecrease in fluorescence, whereas binding with an incorrect nucleotidecauses an increase in fluorescence. Binding between a polymerase and atemplate nucleic acid in the presence of a correct nucleotide may inducean increase in fluorescence, whereas binding with an incorrectnucleotide causes a decrease in fluorescence. The fluorescent signalsmay be used to monitor the kinetics of a nucleotide-inducedconformational change and identify the next base in the template nucleicacid sequence.

Optionally, the crippled DNA polymerase/nucleic-acid interaction may bemonitored by scattering signal originating from the polymerase or tagsattached to the polymerase, for instance, nanoparticle tags.

Conditions for Forming and Manipulating Closed-Complexes

As used herein, a closed-complex can be a ternary complex that includesa crippled DNA polymerase, primed template nucleic acid, and nucleotide.The closed-complex may be in a pre-chemistry conformation, wherein anucleotide is sequestered but not incorporated. The closed-complex mayalternatively be in a pre-translocation conformation, wherein anucleotide is incorporated by formation of a phosphodiester bond withthe 3′-end of the primer in the primed template nucleic acid. Theclosed-complex may be formed in the absence of catalytic metal ions ordeficient levels of catalytic metal ions, thereby physicallysequestering the next correct nucleotide within the polymerase activesite without chemical incorporation. Optionally, the sequesterednucleotide may be a non-incorporable nucleotide. The closed-complex maybe formed in the presence of catalytic metal ions, where theclosed-complex includes a nucleotide analog which is incorporated, but aPPi is not capable of release. In this instance, the closed-complex isstabilized in a pre-translocation conformation. Optionally, apre-translocation conformation is stabilized by chemically cross-linkingthe polymerase. Optionally, the closed-complex may be stabilized byexternal means. In some instances, the closed-complex may be stabilizedby allosteric binding of small molecules, or macromolecules such asantibodies or aptamers. Optionally, closed-complex may be stabilized bypyrophosphate analogs that bind close to the active site with highaffinity, preventing translocation of the polymerase.

As used herein, a stabilized closed-complex or stabilized ternarycomplex refers to a polymerase trapped at the polymerization initiationsite (3′-end of the primer) of the primed template nucleic acid by oneor a combinations of means, including but not limited to, crosslinkingthe thumb and finger domains in the closed conformation, binding of anallosteric inhibitor that prevents return of the polymerase to an openconformation, binding of pyrophosphate analogs that trap polymerase inthe pre-translocation step, absence of catalytic metal ions in theactive site of the polymerase, and addition of a metal ions such asnickel, tin and Sr²⁺ as substitutes for a catalytic metal ion. As such,the polymerase may be trapped at the polymerization initiation site evenafter the incorporation of a nucleotide. Therefore, the polymerase maybe trapped in the pre-chemistry conformation, pre-translocation step,post-translocation step or any intermediate step thereof. Thus, allowingfor sufficient examination and identification of the next correctnucleotide or base.

As described herein, a polymerase-based, sequencing-by-binding reactiongenerally involves providing a primed template nucleic acid with apolymerase and one or more types of nucleotides, wherein the nucleotidesmay or may not be complementary to the next base of the primed templatenucleic acid, and examining the interaction of the polymerase with theprimed template nucleic acid under conditions wherein either chemicalincorporation of a nucleotide into the primed template nucleic acid isdisabled or severely inhibited in the pre-chemistry conformation or oneor more complementary nucleotide incorporation occurs at the 3′-end ofthe primer. Optionally, wherein the pre-chemistry conformation isstabilized prior to nucleotide incorporation, preferably usingstabilizers, a separate incorporation step may follow the examinationstep to incorporate a single nucleotide to the 3′-end of the primer.Optionally, where a single nucleotide incorporation occurs, thepre-translocation conformation may be stabilized to facilitateexamination and/or prevent subsequent nucleotide incorporation.

As indicated above, the presently described methods for sequencing anucleic acid include an examination step. The examination step involvesbinding a polymerase to the polymerization initiation site of a primedtemplate nucleic acid in a reaction mixture including one or morenucleotides, and monitoring the interaction. Optionally, a nucleotide issequestered within the polymerase-primed template nucleic acid complexto form a closed-complex, under conditions in which incorporation of theenclosed nucleotide by the polymerase is attenuated or inhibited.Optionally a stabilizer is added to stabilize the ternary complex in thepresence of the next correct nucleotide. This closed-complex is in astabilized or polymerase-trapped pre-chemistry conformation. Aclosed-complex allows for the incorporation of the enclosed nucleotidebut does not allow for the incorporation of a subsequent nucleotide.This closed-complex is in a stabilized or trapped pre-translocationconformation. Optionally, the polymerase is trapped at thepolymerization site in its closed-complex by one or a combination ofmeans including, but not limited to, crosslinking of the polymerasedomains, crosslinking of the polymerase to the nucleic acid, allostericinhibition by small molecules, uncompetitive inhibitors, competitiveinhibitors, non-competitive inhibitors, and denaturation; wherein theformation of the trapped closed-complex provides information about theidentity of the next base on the nucleic acid template.

Optionally, a closed-complex is released from its trapped or stabilizedconformation, which may allow for nucleotide incorporation to the 3′-endof the template nucleic acid primer. The closed-complex can bedestabilized and/or released by modulating the composition of thereaction conditions. In addition, the closed-complex can be destabilizedby electrical, magnetic, and/or mechanical means. Mechanical meansinclude mechanical agitation, for example, by using ultrasoundagitation. Mechanical vibration destabilizes the closed-complex andsuppresses binding of the polymerase to the DNA. Thus, rather than awash step where the examination reaction mixture is replaced with anincorporation mixture, mechanical agitation may be used to remove thepolymerase from the template nucleic acid, enabling cycling throughsuccessive incorporation steps with a single nucleotide addition perstep.

Any combination of closed-complex stabilization or closed-complexrelease reaction conditions and/or methods may be combined. For example,a polymerase inhibitor that stabilizes a closed-complex may be presentin the examination reaction with a catalytic ion, which functions torelease the closed-complex. In the aforementioned example, theclosed-complex may be stabilized or released, depending on thepolymerase inhibitor properties and concentration, the concentration ofthe catalytic metal ion, other reagents and/or conditions of thereaction mixture, and any combination thereof.

The closed-complex can be stabilized under reaction conditions wherecovalent attachment of a nucleotide to the 3′-end of the primer in theprimed template nucleic acid is attenuated. Optionally, theclosed-complex is in a pre-chemistry conformation or ternary complex.Optionally, the closed-complex is in a pre-translocation conformation.The formation of this closed-complex can be initiated and/or stabilizedby modulating the availability of a catalytic metal ion that permitsclosed-complex release and/or chemical incorporation of a nucleotide tothe primer in the reaction mixture. Exemplary metal ions include, butare not limited to, magnesium, manganese, cobalt, and barium. Catalyticions may be any formulation, for example, salts such as MgCl₂,Mg(CH₃CO₂)₂, and MnCl₂.

The selection and/or concentration of the catalytic metal ion may bebased on the polymerase and/or nucleotides in the sequencing reaction.For example, the HIV reverse transcriptase utilizes magnesium fornucleotide incorporation (N Kaushik, Biochemistry 35:11536-11546 (1996),and H P Patel, Biochemistry 34:5351-5363 (1995), which are incorporatedby reference herein in their entireties). The rate of closed-complexformation using magnesium versus manganese can be different depending onthe polymerase and the identity of the nucleotide. Thus, the stabilityof the closed-complex may differ depending on catalytic metal ion,polymerase, and/or nucleotide identity. Further, the concentration ofcatalytic ion necessary for closed-complex stabilization may varydepending on the catalytic metal ion, polymerase, and/or nucleotideidentity and can be readily determined using the guidance providedherein. For example, nucleotide incorporation may occur at highcatalytic ion concentrations of one metal ion but does not occur at lowconcentrations of the same metal ion, or vice versa. Therefore,modifying metal ion identity, metal ion concentration, polymeraseidentity, and/or nucleotide identity allows for controlled examinationreaction conditions.

The closed-complex may be formed and/or stabilized by sequestering,removing, reducing, omitting, and/or chelating a catalytic metal ionduring the examination step of the sequencing reaction so thatclosed-complex release and/or chemical incorporation does not occur.Chelation includes any procedure that renders the catalytic metal ionunavailable for nucleotide incorporation, including using EDTA and/orEGTA. A reduction includes diluting the concentration of a catalyticmetal ion in the reaction mixture. The reaction mixture can be dilutedor replaced with a solution including a non-catalytic ion, which permitsclosed-complex formation, but inhibits nucleotide incorporation.Non-catalytic ions include, but are not limited to, calcium, strontium,scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper,zinc, gallium, germanium, arsenic, selenium, rhodium, and strontium.Optionally, Ni²⁺ is provided in an examination reaction to facilitateclosed-complex formation. Optionally, Sr²⁺ is provided in an examinationreaction to facilitate closed-complex formation. Optionally, anon-catalytic metal ion that inhibits polymerase-mediated incorporationand a catalytic metal ion are both present in the reaction mixture,wherein one ion is present in a higher effective concentration than theother. In the provided methods, a non-catalytic ion such as cobalt canbecome catalytic (i.e., facilitate nucleotide incorporation) at highconcentrations. Thus, optionally, a low concentration of a non-catalyticmetal ion that inhibits polymerase-mediated incorporation is used tofacilitate ternary complex formation, and a higher concentration of thenon-catalytic metal ion is used to facilitate incorporation.

Non-catalytic ions may be added to a reaction mixture under examinationconditions. The reaction may already include nucleotides. Optionally,non-catalytic ions are complexed to one or more nucleotides andcomplexed nucleotides are added to the reaction mixture. Non-catalyticions can complex to nucleotides by mixing nucleotides with non-catalyticions at elevated temperatures (about 80° C.). For example, a chromiumnucleotide complex may be added to a mixture to facilitateclosed-complex formation and stabilization. Optionally, a chromiumnucleotide complex is a chromium monodentate, bidentate, or tridentatecomplex. Optionally, a chromium nucleotide complex is an α-monodentate,or β-γ-bidentate nucleotide.

Optionally, a closed-complex is formed between a crippled DNApolymerase, primed template nucleic acid, and nucleotide in reactionconditions including Sr²⁺, wherein Sr²⁺ promotes the formation of theclosed-complex. The presence of Sr²⁺ can allow for the favorableformation of a closed-complex including a next correct nucleotide overthe formation a complex including an incorrect nucleotide. The Sr²⁺ ionmay be present at concentrations from about 0.01 mM to about 30 mM.Optionally, Sr²⁺ is present as 10 mM SrCl₂. The formation of theclosed-complex is monitored under examination conditions to identify thenext base in the template nucleic acid of the closed-complex. Theaffinity of the polymerase (e.g., Klenow fragment of E. coli DNApolymerase I, Bst) for each of the dNTPs (e.g., dATP, dTTP, dCTP, dGTP)in the presence of Sr²⁺ can be different. Therefore, examination caninvolve measuring the binding affinities of polymerase-template nucleicacids to dNTPs; wherein binding affinity is indicative of the next basein the template nucleic acid. Optionally, the binding interaction may beperformed under conditions that destabilize the binary interactionsbetween the crippled DNA polymerase and primed template nucleic acid.Optionally, the binding interaction may be performed under conditionsthat stabilize the ternary interactions between the polymerase, theprimed template nucleic acid, and the next correct nucleotide. Afterexamination, a wash step removes unbound nucleotides, and Mg²⁺ is addedto the reaction to induce pyrophosphate (PPi) cleavage and nucleotideincorporation. Optionally, the wash step includes Sr²⁺ to maintain thestability of the ternary complex, preventing the dissociation of theternary complex. The reaction may be repeated until a desired sequenceread-length is obtained.

Optionally, a closed-complex is formed between a crippled DNApolymerase, primed template nucleic acid, and nucleotide in reactionconditions including Ni²⁺, wherein Ni²⁺ promotes the formation of theclosed-complex. The presence of Ni²⁺ can allow for the favorableformation of a closed-complex including a next correct nucleotide overthe formation a complex including an incorrect nucleotide. The Ni²⁺ ionmay be present at concentrations from about 0.01 mM to about 30 mM.Optionally, Ni²⁺ is present as 10 mM NiCl₂. The formation of theclosed-complex is monitored under examination conditions to identify thenext base in the template nucleic acid of the closed-complex. Theaffinity of the polymerase (e.g., Klenow fragment of E. coli DNApolymerase I, Bst) for each of the dNTPs (e.g., dATP, dTTP, dCTP, dGTP)in the presence of Sr²⁺ can be different. Therefore, examination caninvolve measuring the binding affinities of polymerase-template nucleicacids to dNTPs; wherein binding affinity is indicative of the next basein the template nucleic acid. Optionally, the binding interaction may beperformed under conditions that destabilize the binary interactionsbetween the polymerase and primed template nucleic acid. Optionally, thebinding interaction may be performed under conditions that stabilize theternary interactions between the polymerase, the primed template nucleicacid, and the next correct nucleotide. After examination, a wash removesunbound nucleotides and polymerase, and Mg²⁺ is added to the reaction toinduce pyrophosphate (PPi) cleavage and nucleotide incorporation.Optionally, the wash buffer includes Ni²⁺ to maintain the stability ofthe ternary complex, preventing the dissociation of the ternary complex.The reaction may be repeated until a desired sequence read length isobtained.

Optionally, a closed-complex is formed between a crippled DNApolymerase, primed template nucleic acid, and nucleotide in reactionconditions including non-catalytic concentrations of Co²⁺, wherein Co²⁺promotes the formation of the closed-complex. The presence ofnon-catalytic concentrations of Co²⁺ can allow for the favorableformation of a closed-complex including a next correct nucleotide overthe formation a complex including an incorrect nucleotide. The Co²⁺ ionmay be present at concentrations from about 0.01 mM to about 0.5 mM.Optionally, Co²⁺ is present as 0.5 mM CoCl₂. The formation of theclosed-complex is monitored under examination conditions to identify thenext base in the template nucleic acid of the closed-complex. Theaffinity of the polymerase (e.g., Klenow fragment of E. coli DNApolymerase I, Bst) for each of the dNTPs (e.g., dATP, dTTP, dCTP, dGTP)in the presence of Co²⁺ can be different. Therefore, examination caninvolve measuring the binding affinities of polymerase-template nucleicacids to dNTPs; wherein binding affinity is indicative of the next basein the template nucleic acid. Optionally, the binding interaction may beperformed under conditions that destabilize the binary interactionsbetween the polymerase and primed template nucleic acid. Optionally, thebinding interaction may be performed under conditions that stabilize theternary interactions between the polymerase, the primed template nucleicacid, and the next correct nucleotide. After examination, a wash removesunbound nucleotides and polymerase, and Co²⁺ at a catalyticconcentration is added to the reaction to induce pyrophosphate (PPi)cleavage and nucleotide incorporation. Optionally, the wash bufferincludes non-catalytic amounts of Co²⁺ to maintain the stability of theternary complex, preventing the dissociation of the ternary complex. Thereaction may be repeated until a desired sequence read length isobtained.

Optionally, a catalytic metal ion may facilitate the formation of aclosed-complex without subsequent nucleotide incorporation andclosed-complex release. Optionally, a concentration of 0.5, 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 μM Mg²⁺ in a reaction mixture can induceconformational change of a polymerase to form a closed-complex withoutsubsequent nucleotide incorporation, PPi and closed-complex release.Optionally, the concentration of Mg²⁺ is from about 0.5 μM to about 10μM, from about 0.5 μM to about 5 μM, from about 0.5 μM to about 4 μM,from about 0.5 μM to about 3 μM, from about μM to about 5 μM, from about1 μM to about 4 μM, and from about 1 μM to about 3 μM.

Optionally, the concentration of available catalytic metal ion in thesequencing reaction which is necessary to allow nucleotide incorporationis from about 0.001 mM to about 10 mM, from about 0.01 mM to about 5 mM,from about 0.01 mM to about 3 mM, from about 0.01 mM to about 2 mM, fromabout 0.01 mM to about 1 mM, from about 0.05 mM to about 10 mM, fromabout 0.05 mM to about 5 mM, from about 0.05 mM to about 3 mM, fromabout 0.05 to about 2 mM, or from about 0.05 mM to about 1 mM.Optionally, the concentration of catalytic metal ion is from 5 mM to 50mM. Optionally, the concentration of catalytic metal ion is from 5 mM to15 mM, or about 10 mM.

A non-catalytic ion may be added to the reaction mixture at any stageincluding before, during, or after any of the following reaction steps:providing a primed template nucleic acid, providing a polymerase,formation of a binary complex, providing a nucleotide, formation of apre-chemistry closed-complex, nucleotide incorporation, formation of apre-translocation closed-complex, and formation of a post-translocationconformation. The non-catalytic ion may be added to the reaction mixtureduring wash steps. The non-catalytic ion may be present through thereaction in the reaction mixture. For example, a catalytic ion is addedto the reaction mixture at concentrations which dilute the non-catalyticmetal ion that inhibits polymerase-mediated incorporation, allowing fornucleotide incorporation.

The ability of catalytic and non-catalytic ions to modulate nucleotideincorporation may depend on conditions in the reaction mixtureincluding, but not limited to, pH, ionic strength, chelating agents,chemical cross-linking, modified polymerases, non-incorporablenucleotides, mechanical or vibration energy, and electric fields.

Optionally, the concentration of non-catalytic metal ion that inhibitspolymerase-mediated incorporation in the sequencing reaction necessaryto allow for closed-complex formation without nucleotide incorporationis from about 0.1 mM to about 50 mM, from about 0.1 mM to about 40 mM,from about 0.1 mM to about 30 mM, from about 0.1 mM to about 20 mM, fromabout 0.1 mM to about 10 mM, from about 0.1 mM to about 5 mM, from about0.1 to about 1 mM, from about 1 mM to about 50 mM, from about 1 to about40 mM, from about 1 mM to about 30 mM, from about 1 mM to about 20 mM,from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, fromabout 2 mM to about 30 mM, from about 2 mM to about 20 mM, from about 2mM to about 10 mM, or any concentration within these ranges.

A closed-complex may be formed and/or stabilized by the addition of apolymerase inhibitor to the examination reaction mixture. Inhibitormolecules phosphonoacetate (phosphonoacetic acid) and phosphonoformate(phosphonoformic acid, common name Foscarnet), Suramin, Aminoglycosides,INDOPY-1 and Tagetitoxin are non-limiting examples of uncompetitive ornoncompetitive inhibitors of polymerase activity. The binding of theinhibitor molecule, near the active site of the enzyme, traps thepolymerase in either a pre-translocation or post-translocation step ofthe nucleotide incorporation cycle, stabilizing the polymerase in itsclosed-complex conformation before or after the incorporation of anucleotide, and forcing the polymerase to be bound to the templatenucleic acid until the inhibitor molecules are not available in thereaction mixture by removal, dilution or chelation.

Thus, provided is a method for sequencing a template nucleic acidmolecule including an examination step including providing a templatenucleic acid molecule primed with a primer; contacting the primedtemplate nucleic acid molecule with a first reaction mixture including apolymerase, a polymerase inhibitor and at least one unlabeled nucleotidemolecule; monitoring the interaction of the polymerase with the primedtemplate nucleic acid molecule in the presence of the unlabelednucleotide molecule without incorporation of the nucleotide into theprimer of the primed template nucleic acid molecule; and identifying thenucleotide that is complementary to the next base of the primed templatenucleic acid molecule by the monitored interaction. The polymeraseinhibitor prevents the incorporation of the unlabeled nucleotidemolecule into the primer of the primer template nucleic acid.Optionally, the inhibitor is a non-competitive inhibitor, an allostericinhibitor, or an uncompetitive allosteric inhibitor. Optionally, thepolymerase inhibitor competes with a catalytic ion binding site in thepolymerase.

Detection Platforms: Instrumentation for Detecting the Closed-Complex

The interaction between the crippled DNA polymerase and the templatenucleic acid in the presence of nucleotides can be monitored without theuse of an exogenous label. For example, the sequencing reaction may bemonitored by detecting the change in refractive index, fluorescenceemission, charge detection, Raman scattering detection, ellipsometrydetection, pH detection, size detection, mass detection, surface plasmonresonance, guided mode resonance, nanopore optical interferometry,whispering gallery mode resonance, nanoparticle scattering, photoniccrystal, quartz crystal microbalance, bio-layer interferometry,vibrational detection, pressure detection and other label-free detectionschemes that detect the added mass or refractive index due to polymerasebinding in a closed-complex with a template nucleic acid.

Optionally, detecting a change in refractive index is accomplished byone or a combination of means, including, but not limited to, surfaceplasmon resonance sensing, localized plasmon resonance sensing,plasmon-photon coupling sensing, transmission sensing throughsub-wavelength nanoholes (enhanced optical transmission), photoniccrystal sensing, interferometry sensing, refraction sensing, guided moderesonance sensing, ring resonator sensing, or ellipsometry sensing.Optionally, nucleic acid molecules may be localized to a surface,wherein the interaction of polymerase with nucleic acids in the presenceof various nucleotides may be measured as a change in the localrefractive index.

Optionally, the template nucleic acid is tethered to or localizedappropriately on or near a surface, such that the interaction ofpolymerase and template nucleic acid in the presence of nucleotideschanges the light transmitted across or reflected from the surface. Thesurface may contain nanostructures. Optionally, the surface is capableof sustaining plasmons or plasmon resonance. Optionally, the surface isa photonic substrate, not limited to a resonant cavity, resonant ring orphotonic crystal slab. Optionally, the surface is a guided moderesonance sensor. Optionally, the nucleic acid is tethered to, orlocalized appropriately on or near a nanohole array, a nanoparticle or amicroparticle, such that the interaction of polymerase and templatenucleic acid in the presence of nucleotides changes the absorbance,scattering, reflection or resonance of the light interacting with themicroparticle or nanoparticle.

Optionally, a nanohole array on a gold surface is used as a refractiveindex sensor. The template nucleic acid may be attached to a metalsurface by standard thiol chemistry, incorporating the thiol group onone of the primers used in a PCR reaction to amplify the DNA. When thedimensions of the nanohole array are appropriately tuned to the incidentlight, binding of the polymerase to the template nucleic acid in thepresence of nucleotides can be monitored as a change in lighttransmitted across the nanoholes. For both the labeled and label-freeschemes, simple and straightforward measurement of equilibrium signalintensity may reveal the formation of a stable closed-complex.

Optionally, nucleic acid molecules are localized to a surface capable ofsustaining surface plasmons, wherein the change in refractive indexcaused by the polymerase interaction with localized nucleic acids may bemonitored through the change in the properties of the surface plasmons,wherein further, said properties of surface plasmons may include surfaceplasmon resonance. Surface plasmons, localized surface plasmons (LSP),or surface plasmon polaritons (SPP), arise from the coupling ofelectromagnetic waves to plasma oscillations of surface charges. LSPsare confined to nanoparticle surfaces, while SPPs and are confined tohigh electron density surfaces, at the interface between high electronmobility surfaces and dielectric media. Surface plasmons may propagatealong the direction of the interface, whereas they penetrate into thedielectric medium only in an evanescent fashion. Surface plasmonresonance conditions are established when the frequency of incidentelectromagnetic radiation matches the natural frequency of oscillationof the surface electrons. Changes in dielectric properties at theinterface, for instance due to binding or molecular crowding, affectsthe oscillation of surface electrons, thereby altering the surfaceplasmon resonance wavelength. Surfaces capable of surface plasmonresonance include, in a non-limiting manner, nanoparticles, clusters andaggregates of nanoparticles, continuous planar surfaces, nanostructuredsurfaces, and microstructured surfaces. Materials such as gold, silver,aluminum, high conductivity metal oxides (e.g., indium tin oxide, zincoxide, tungsten oxide) are capable of supporting surface plasmonresonance at their surfaces.

Optionally, a single nucleic acid molecule, or multiple clonal copies ofa nucleic acid, are attached to a nanoparticle, such that binding ofpolymerase to the nucleic acid causes a shift in the localized surfaceplasmon resonance (LSPR). Light incident on the nanoparticles inducesthe conduction electrons in them to oscillate collectively with aresonant frequency that depends on the nanoparticles' size, shape andcomposition. Nanoparticles of interest may assume different shapes,including spherical nanoparticles, nanorods, nanopyramids, nanodiamonds,and nanodiscs. As a result of these LSPR modes, the nanoparticles absorband scatter light so intensely that single nanoparticles are easilyobserved by eye using dark-field (optical scattering) microscopy. Forexample, a single 80-nm silver nanosphere scatters 445-nm blue lightwith a scattering cross-section of 3×10⁻² m², a million-fold greaterthan the fluorescence cross-section of a fluorescein molecule, and athousand fold greater than the cross-section of a similarly sizednanosphere filled with fluorescein to the self-quenching limit.Optionally, the nanoparticles are plasmon-resonant particles configuredas ultra-bright, nanosized optical scatters with a scattering peakanywhere in the visible spectrum. Plasmon-resonant particles areadvantageous as they do not bleach. Optionally, plasmon-resonantparticles are prepared, coated with template nucleic acids, and providedin a reaction mixture including a polymerase and one or morenucleotides, wherein a polymerase-template nucleic acid-particleinteraction is detected. One or more of the aforementioned steps may bebased on or derived from one or more methods disclosed by Schultz etal., in PNAS 97:996-1001 (2000), which is incorporated by referenceherein in its entirety.

The very large extinction coefficients at resonant wavelength enablesnoble-metal nanoparticles to serve as extremely intense labels fornear-surface interactions. Optionally, polymerase interaction withnanoparticle-localized DNA results in a shift in the resonantwavelength. The change in resonant wavelength due to binding or bindinginteractions can be measured in one of many ways. Optionally, theillumination is scanned through a range of wavelengths to identify thewavelength at which maximum scattering is observed at an imaging device.Optionally, broadband illumination is utilized in conjunction with adispersive element near the imaging device, such that the resonant peakis identified spectroscopically. Optionally, the nanoparticle system maybe illuminated at its resonant wavelength, or near its resonantwavelength, and any binding interactions may be observed as a drop inintensity of light scattered as the new resonant wavelength shifts awayfrom the illumination wavelength. Depending on the positioning of theilluminating wavelength, interactions may even appear as an increase innanoparticle scattering as the resonance peak shifts towards theillumination wavelength. Optionally, DNA-attached-nanoparticles may belocalized to a surface, or, alternatively, theDNA-attached-nanoparticles may be suspended in solution. A comprehensivereview of biosensing using nanoparticles is described by Anker et al.,in Nature Materials 7: 442-453 (2008), which is incorporated in itsentirety herein by reference.

Optionally, nano-features capable of LSPR are lithographically patternedon a planar substrate. The two dimensional patterning of nano-featureshas advantages in multiplexing and high-throughput analysis of a largenumber of different nucleic acid molecules. Optionally, gold nanopostsare substrates for surface plasmon resonance imaging detection ofpolymerase-template nucleic acid interactions, wherein the nucleic acidsare attached to the nanoposts. Nanostructure size and period caninfluence surface plasmon resonance signal enhancement, optionally,providing a 2, 3, 4, 5, 6, 7, 8-fold or higher signal amplification whencompared to control films.

Optionally, surface plasmon resonance may be sustained in planarsurfaces. A number of commercial instruments based on the Kretschmannconfiguration (e.g., Biacore, Uppsala, Sweden) and surface plasmonresonance imaging (e.g., GWC Technologies; Madison, Wis.; or Horiba;Kyoto, Japan) are available and have well established protocols forattaching DNA to their surfaces, as single spots and in multiplexedarray patterns. In the Kretschmann configuration, a metal film,typically gold, is evaporated onto the side of a prism and incidentradiation is launched at an angle to excite the surface plasmons. Anevanescent wave penetrates through the metal film exciting plasmons onthe other side, where it may be used to monitor near-surface and surfaceinteractions near the gold film. At the resonant angle, the lightreflected from the prism-gold interface is severely attenuated. Assumingfixed wavelength illumination, binding interactions may be examined bymonitoring both the intensity of the reflected light at a fixed angleclose to the resonant angle, as well as by monitoring the changes inangle of incidence required to establish surface plasmon resonanceconditions (minimum reflectivity). When a 2D imaging device such as aCCD or CMOS camera is utilized to monitor the reflected light, theentire illumination area may be imaged with high resolution. This methodis called surface plasmon resonance imaging (SPRi). It allows highthroughput analysis of independent regions on the surfacesimultaneously. Broadband illumination may also be used, in a fixedangle configuration, wherein the wavelength that is coupled to thesurface plasmon resonance is identified spectroscopically by looking fordips in the reflected spectrum. Surface interactions are monitoredthrough shifts in the resonant wavelength.

Surface plasmon resonance is a well-established method for monitoringprotein-nucleic acid interactions, and there exist many standardprotocols both for nucleic acid attachment as well as for analyzing thedata. Illustrative references from the literature include Cho et al.,“Binding Kinetics of DNA-Protein Interaction Using Surface PlasmonResonance,” Protocol Exchange, May 22, 2013; and Brockman et al., “AMultistep Chemical Modification Procedure To Create DNA Arrays on GoldSurfaces for the Study of Protein-DNA Interactions with Surface PlasmonResonance Imaging,” Journal of the American Chemical Society 121:8044-51 (1999), both of which are incorporated by reference herein intheir entireties.

Polymerase/nucleic-acid interactions may be monitored on nanostructuredsurfaces capable of sustaining localized surface plasmons. Optionally,polymerase/nucleic-acid interactions may be monitored on nanostructuredsurfaces capable of sustaining surface plasmon polaritons.

Optionally, polymerase/nucleic-acid interactions may be monitored onnanostructured surfaces capable of sustaining localized surfaceplasmons. Optionally, polymerase/nucleic-acid interactions may bemonitored on nanostructured surfaces capable of sustaining surfaceplasmon polaritons.

Optionally, extraordinary optical transmission (EOT) through a nanoholesarray may be used to monitor nucleic-acid/polymerase interactions. Lighttransmitted across subwavelength nanoholes in plasmonic metal films ishigher than expected from classical electromagnetic theory. Thisenhanced optical transmission may be explained by considering plasmonicresonant coupling to the incident radiation, whereby at resonantwavelength, a larger than anticipated fraction of light is transmittedacross the metallic nanoholes. The enhanced optical transmission isdependent on the dimensions and pitch of the nanoholes, properties ofthe metal, as well as the dielectric properties of the medium on eitherside of the metal film bearing the nanoholes. In the context of abiosensor, the transmissivity of the metallic nanohole array depends onthe refractive index of the medium contacting the metal film, whereby,for instance, the interaction of polymerase with nucleic acid attachedto the metal surface may be monitored as a change in intensity of lighttransmitted across the nanoholes array. Instrumentation and alignmentrequirements when using the EOT/plasmonic nanohole array approach ofsurface plasmon resonance may be employed using very compact optics andimaging elements. Low power LED illumination and a CMOS or CCD cameramay suffice to implement robust EOT plasmonic sensors. An exemplarynanohole array-based surface plasmon resonance sensing device isdescribed by Escobedo et al., in “Integrated Nanohole Array SurfacePlasmon Resonance Sensing Device Using a Dual-Wavelength Source,”Journal of Micromechanics and Microengineering 21: 115001 (2011), whichis herein incorporated by reference in its entirety.

The plasmonic nanohole array may be patterned on an optically opaquelayer of gold (greater than 50 nm thickness) deposited on a glasssurface. Optionally, the plasmonic nanohole array may be patterned on anoptically thick film of aluminum or silver deposited on glass.Optionally, the nanohole array is patterned on an optically thick metallayer deposited on low refractive index plastic. Patterning plasmonicnanohole arrays on low refractive index plastics enhances thesensitivity of the device to refractive index changes by better matchingthe refractive indices on the two sides of the metal layer. Optionally,refractive index sensitivity of the nanohole array is increased byincreasing the distance between holes. Optionally, nanohole arrays arefabricated by replication, for example, by embossing, casting,imprint-lithography, or template-stripping. Optionally, nanohole arraysare fabricated by self-assembly using colloids. Optionally, nanoholearrays are fabricated by projection direct patterning, such as laserinterference lithography.

A nano-bucket configuration may be preferable to a nanoholeconfiguration. In the nanohole configuration, the bottom of thenano-feature is glass or plastic or other appropriate dielectric,whereas in the nano-bucket configuration, the bottom of the nano-featureincludes a plasmonic metal. The nano-bucket array advantageously isrelatively simple to fabricate while maintaining the transmissionsensitivity to local refractive index.

Optionally, the nanohole array plasmonic sensing is combined withlens-free holographic imaging for large area imaging in an inexpensivemanner. Optionally, a plasmonic biosensing platform includes a plasmonicchip with nanohole arrays, a light-emitting diode source configured toilluminate the chip, and a CMOS imager chip to record diffractionpatterns of the nanoholes, which is modulated by molecular bindingevents on the surface. The binding events may be the formation of aclosed-complex between a polymerase and a template nucleic acid in thepresence of a nucleotide.

The methods to functionalize surfaces (for nucleic acid attachment) forsurface plasmon resonance sensing may be directly applied to EOTnanohole arrays as both sensing schemes employ similar metal surfaces towhich nucleic acids need to be attached.

Optionally, the refractive index changes associated withpolymerase/nucleic acid interaction may be monitored on nanostructuredsurfaces that do not support plasmons. Optionally, guided mode resonancemay be used to monitor the polymerase/nucleic-acid interaction.Guided-mode resonance or waveguide-mode resonance is a phenomenonwherein the guided modes of an optical waveguide can be excited andsimultaneously extracted by the introduction of a phase-matchingelement, such as a diffraction grating or prism. Such guided modes arealso called “leaky modes,” as they do not remain guided and have beenobserved in one and two-dimensional photonic crystal slabs. Guided moderesonance may be considered a coupling of a diffracted mode to awaveguide mode of two optical structured placed adjacent or on top ofeach other. For instance, for a diffraction grating placed on top of anoptical waveguide, one of the diffracted modes may couple exactly intothe guided mode of the optical waveguide, resulting in propagation ofthat mode along the waveguide. For off-resonance conditions, no light iscoupled into the waveguide, so the structure may appear completelytransparent (if dielectric waveguides are used). At resonance, theresonant wavelength is strongly coupled into the waveguide and may becouple out of the structure depending on downstream elements from thegrating-waveguide interface. In cases where the grating coupler isextended over the entire surface of the waveguide, the light cannot beguided, as any light coupled in is coupled out at the next gratingelement. Therefore, in a grating waveguide structure, resonance isobserved as a strong reflection peak, whereas the structure istransparent to off-resonance conditions. The resonance conditions aredependent on angle, grating properties, polarization and wavelength ofincident light. For cases where the guided mode propagation is notpresent, for instance due to a grating couple to the entire surface ofthe waveguide, the resonant mode may also be called leaky-moderesonance, in light of the strong optical confinement and evanescentpropagation of radiation in a transverse direction from the waveguidelayer. Change in dielectric properties near the grating, for instancedue to binding of biomolecules affects the coupling into the waveguide,thereby altering the resonant conditions. Optionally, where nucleic acidmolecules are attached to the surface of grating waveguide structures,the polymerase/nucleic-acid interaction may be monitored as a change inwavelength of the leaky mode resonance.

A diffraction element may be used directly on a transparent substratewithout an explicit need for a waveguide element. The change inresonance conditions due to interactions near the grating nanostructuremay be monitored as resonant wavelength shifts in the reflected ortransmitted radiation.

Reflected light from a nucleic acid attached guided mode resonant sensormay be used to monitor the polymerase/nucleic-acid interaction. Abroadband illumination source may be employed for illumination, and aspectroscopic examination of reflected light could reveal changes inlocal refractive index due to polymerase binding.

Optionally, a broadband illumination may be used and the transmittedlight may be examined to identify resonant shifts due to polymeraseinteraction. A linearly polarized narrow band illumination may be used,and the transmitted light may be filtered through a cross-polarizer;wherein the transmitted light is completely attenuated due to thecrossed polarizers excepting for the leaky mode response whosepolarization is modified. This implementation converts refractive indexmonitoring to a simple transmission assay that may be monitored oninexpensive imaging systems. Published material describe the assembly ofthe optical components. See, Nazirizadeh et al., “Low-Cost Label-FreeBiosensors Using Photonic Crystals Embedded between Crossed Polarizers,”Optics Express 18: 19120-19128 (2010), which is incorporated herein byreference in its entirety.

In addition to nanostructured surfaces, plain, unstructured surfaces mayalso be used advantageously for monitoring refractive index modulations.Optionally, interferometry may be employed to monitor the interaction ofpolymerase with nucleic acid bound to an un-structured, opticallytransparent substrate. Nucleic acid molecules may be attached to the topsurface of a glass slide by any means known in the art, and the systemilluminated from the bottom surface of the glass slide. There are tworeflection surfaces in this configuration, one reflection from thebottom surface of the glass slide, and the other from the top surfacewhich has nucleic acid molecules attached to it. The two reflected wavesmay interfere with each other causing constructive or destructiveinterference based on the path length differences, with the wavereflected from the top surface modulated by the changes in dielectricconstant due to the bound nucleic acid molecules (and subsequently bythe interaction of polymerase with the bound nucleic acid molecules).With the reflection from the bottom surface unchanged, any binding tothe nucleic acid molecules may be reflected in the phase differencebetween the beams reflected from the top and bottom surfaces, which inturn affects the interference pattern that is observed. Optionally,bio-layer interferometry is used to monitor the nucleic acid/polymeraseinteraction. Bio-layer interferometry may be performed on commercialdevices such as those sold by Pall Forte Bio corporation (Menlo Park,Calif.).

Optionally, the reflected light from the top surface is selectivelychosen by using focusing optics. The reflected light from the bottomsurface is disregarded because it is not in the focal plane. Focusingonly on the nucleic-acid-attached top surface, the light collected bythe focusing lens includes a planar wave, corresponding to the partiallyreflected incident radiation, and a scattered wave, corresponding to theradiations scattered in the collection direction by molecules in thefocal plane. These two components may be made to interfere if theincident radiation is coherent. This scattering based interferometricdetection is extremely sensitive and can be used to detect down tosingle protein molecules.

Optionally, a field-effect transistor (FET) is configured as a biosensorfor the detection of a closed-complex. A gate terminal of the FET ismodified by the addition of template nucleic acids. The binding of apolymerase including a charged tag results in changes in electrochemicalsignals. Binding of a polymerase with a next correct nucleotide to thetemplate nucleic acid provides different signals than polymerase bindingto a template nucleic acid in the presence of other incorrectnucleotides, where each incorrect nucleotide may also provide adifferent signal. Optionally, polymerase interactions with a templatenucleic acid are monitored using FET without the use of a an exogenouslabel on the polymerase, primed template nucleic acid, or nucleotide.Optionally, the pH change that occurs due to release of H⁺ ions duringthe incorporation reaction is detected using a FET. Optionally, thepolymerase includes a tag that generates continuous H⁺ ions that isdetected by the FET. Optionally, the continuous H⁺ ion generating tag isan ATP synthase. Optionally, the continuous H⁺ ion generation tag ispalladium, copper or another catalyst. Optionally, the release of a PPiafter nucleotide incorporation is detected using FET. For example, onetype of nucleotide may be provided to a reaction at a time. Once thenext correct nucleotide is added and conditions allow for incorporation,PPi is cleaved, released, and detected using FET, therefore identifyingthe next correct nucleotide and the next base. Optionally, templatenucleic acids are bound to walls of a nanotube. Optionally, a polymeraseis bound to a wall of a nanotube. FET is advantageous for use as asequencing sensor due to its small size and low weight, making itappropriate for use as a portable sequencing monitoring component.Details of FET detection of molecular interactions are described by Kimet al., in “An FET-Type Charge Sensor for Highly Sensitive Detection ofDNA Sequence,” Biosensors and Bioelectronics, Microsensors andMicrosystems 20: 69-74 (2004), doi:10.1016/j.bios.2004.01.025; and byStar et al., in “Electronic Detection of Specific Protein Binding UsingNanotube FET Devices,” Nano Letters 3: 459-63 (2003),doi:10.1021/n10340172, which are incorporated by reference herein intheir entireties.

Optionally, the crippled DNA polymerase includes a fluorescent tag. Tomonitor polymerase-nucleic acid interaction with high signal-to-noise,evanescent illumination or confocal imaging may be employed. Theformation of a closed-complex on localized template nucleic acids may beobserved as an increased fluorescence compared to the background, forinstance, whereas in some instances it may be also be observed as adecreased fluorescence due to quenching or change in local polarenvironment. Optionally, a fraction of polymerase molecules may betagged with a fluorophore while another fraction may be tagged with aquencher in the same reaction mixture; wherein, the formation ofclosed-complex on a localized, clonal population of nucleic acid isrevealed as decrease in fluorescence compared to the background. Theclonal population of nucleic acids may be attached to a support surfacesuch as a planar substrate, microparticle, or nanoparticle. Optionally,a polymerase is tagged with a fluorophore, luminophore,chemiluminophore, chromophore, or bioluminophore. A ternary complex thatincludes a primed template nucleic acid molecule, a cognate nucleotide,and a fluorescently tagged or labeled polymerase can be detected ormonitored by detecting the fluorescent label moiety attached to thepolymerase.

Optionally, a plurality of template nucleic acids is tethered to asurface and one (or more) dNTPs are flowed in sequentially. The spectrumof affinities reveals the identity of the next correct nucleotide andtherefore the next base in the template nucleic acid. Optionally, theaffinities are measured without needing to remove and replace reactionmixture conditions (i.e., a wash step). Autocorrelation of the measuredintensities of the binding interaction, for instance, could readilyreveal the dynamics of nucleic acid sequence. Optionally, examinationincludes monitoring the affinity of the polymerase to the primedtemplate nucleic acid in the presence of nucleotides. Optionally, thepolymerase binds transiently with the nucleic acid and the bindingkinetics and affinity provides information about the identity of thenext base on the template nucleic acid. Optionally, a closed-complex isformed, wherein the reaction conditions involved in the formation of theclosed-complex provide information about the next base on the nucleicacid. Optionally, the polymerase is trapped at the polymerization sitein its the interaction, thus revealing the affinities without requiringa washing step to measure the off-rate.

Any technique that can measure dynamic interactions between a crippledDNA polymerase and nucleic acid may be used to measure the affinitiesand enable the sequencing reaction methods disclosed herein.

Systems for Detecting Nucleotide-Specific Ternary Complex Formation

The provided methods can be performed using a platform, where anycomponent of the nucleic acid polymerization reaction is localized to asurface. Optionally, the template nucleic acid is attached to a planarsubstrate, a nanohole array, a microparticle, or a nanoparticle.Optionally, all reaction components are freely suspended in the reactionmixture, and not immobilized to a solid support substrate.

Optionally, the template nucleic acid is immobilized to a surface. Thesurface may be a planar substrate, a hydrogel, a nanohole array, amicroparticle, or a nanoparticle. Optionally, the reaction mixturescontain a plurality of clonally amplified template nucleic acidmolecules. Optionally, the reaction mixtures contain a plurality ofdistinguishable template nucleic acids.

Provided herein, inter alia, are systems for performing sequencingreactions involving the examination of the interaction between apolymerase and a primed template nucleic acid in the presence ofnucleotides to identify the next base in the template closed-complex byone or a combination of means including, but not limited to,crosslinking of the polymerase domains, crosslinking of the polymeraseto the nucleic acid, allosteric inhibition by small molecules,uncompetitive inhibitors, competitive inhibitors, non-competitiveinhibitors, and denaturation; wherein the formation of the trappedpolymerase complex provides information about the identity of the nextbase on the nucleic acid template.

Also provided is a system for performing one or more steps of anysequencing method disclosed herein. Optionally, the system includescomponents and reagents necessary to perform a polymerase and templatenucleic acid binding assay in the presence of nucleotides, wherein thetemplate nucleic acid is provided on a nanostructure. Optionally, thesystem includes one or more reagents and instructions necessary to bindtemplate DNA molecules onto a nanostructure. For example, the systemprovides a nanostructure, such as a chip, configured for use withsurface plasmon resonance to determine binding kinetics. An example ofsuch a chip is a CM5 Sensor S chip (GE Healthcare; Piscatawany, N.J.).The system may provide instrumentation such as a surface plasmonresonance instrument. The system may provide streptavidin and/or biotin.Optionally, the system provides biotin-DNA, DNA ligase, buffers, and/orDNA polymerase for preparation of biotinylated template DNA. Optionally,the system provides a gel or reagents (e.g., phenol:chloroform) forbiotinylated DNA purification. Alternatively, the system providesreagents for biotinylated template DNA characterization, for example,mass spectrometry or HPLC. Optionally, the system includes streptavidin,a chip, reagents, instrumentation, and/or instructions forimmobilization of streptavidin on a chip. Optionally, a chip is providedin the system already configured for template DNA coating, wherein thechip is immobilized with a reagent capable of binding template nucleicacids or modified template nucleic acids (e.g., biotinylated templateDNA). Optionally, the system provides reagents for chip regeneration.

Also provided is a system for performing one or more steps of anysequencing method disclosed herein. Optionally, the system includescomponents and reagents necessary to perform a polymerase and templatenucleic acid binding assay in the presence of nucleotides, wherein thetemplate nucleic acid is provided on a nanoparticle. Optionally, thesystem includes one or more reagents and instructions necessary to bindtemplate DNA molecules onto a nanoparticle. The nanoparticle may beconfigured for the electrochemical detection of nucleic acid-polymeraseinteraction, for instance, by using gold nanoparticles. Optionally, theDNA-nanoparticle conjugates are formed between aqueous gold colloidsolutions and template DNA molecules including, for example, free thiolor disulfide groups at their ends. The conjugates may include the samenucleic acid sequences. Optionally, the nanoparticle conjugates arestabilized against flocculation and precipitation at high temperature(e.g., greater than 60° C.) and high ionic strength (e.g., 1M Na⁺).Optionally, the system provides reagents for preparing template DNAmolecules for nanoparticle attachment, including, generating templateDNA molecules with disulfides or thiols. Disulfide-containing templatenucleic acids may be synthesized using, for example, a 3′-thiol modifiercontrolled-pore glass (CPG) or by beginning with a universal support CPGand adding a disulfide modifier phosphoramidite as the first monomer inthe sequence. The system may provide nucleic acid synthesis reagentsand/or instructions for obtaining disulfide-modified template nucleicacids. Thiol-containing template nucleic acids may also be generatedduring nucleic acid synthesis with a 5′-tritylthiol modifierphosphoramidite. The system may provide reagents and/or instructions fornanoparticle conjugate purification using for example, electrophoresisor centrifugation. Optionally, nanoparticle conjugates are used tomonitor polymerase-template nucleic acid interactions colorimetrically.In this instance, the melting temperature of the nanoparticle conjugateincreases in the presence of strong polymerase binding. Therefore, thestrength of DNA binding can be determined by the change in this meltingtransition, which is observable by a color change. The systemsoptionally include reagents and equipment for detection of the meltingtransition.

Also provided is a system for performing one or more steps of anysequencing method disclosed herein. Optionally, the system includescomponents and reagents necessary to perform a polymerase and templatenucleic acid binding assay in the presence of nucleotides, using adetectable polymerase. Optionally, the polymerase is detectably labeled.Optionally, the polymerase is detected using intrinsic properties of thepolymerase, for example, aromatic amino acids. Optionally, thepolymerase and template nucleic acids present in the system areconfigured for use in solution, without conjugation to a support. Thedetectable label on the polymerase may be a fluorophore, whereinfluorescence is used to monitor polymerase-template nucleic acid bindingevents. Optionally, the detectable polymerase may be used in combinationwith template nucleic acids in solution, or template nucleic acidsconjugated to a support structure. Optionally, one or more cysteineresidues of the polymerase is labeled with Cy3-maleimide. Optionally,the system includes reagents and/or instructions necessary to preparefluorescently labeled polymerase molecules. The system may includereagents and/or instructions for purification of fluorescently labeledpolymerases.

Procedural Features of the Methods

Following the examination step, where the identity of the next base hasbeen identified via formation of a closed-complex, the reactionconditions may be reset, recharged, or modified as appropriate, inpreparation for the optional incorporation step or an additionalexamination step. Optionally, the identity of the next base has beenidentified without chemically incorporating a nucleotide. Optionally,the identity of the next base is identified with chemical incorporationof a nucleotide, wherein a subsequent nucleotide incorporation has beeninhibited. Optionally, all components of the examination step, excludingthe template nucleic acid being sequenced, are removed or washed away,returning the system to the pre-examination condition. Optionally,partial components of the examination step are removed. Optionally,additional components are added to the examination step.

Optionally, reversible terminator nucleotides are used in theincorporation step to ensure one, and only one nucleotide isincorporated per cycle. No labels are required on the reversibleterminator nucleotides as the base identity is known from theexamination step. Non-fluorescently labeled reversible terminators arereadily available from commercial suppliers. Non-labeled reversibleterminator nucleotides are expected to have much faster incorporationkinetics compared to labeled reversible terminators due to their smallersteric footprint, and similar size to natural nucleotides.

Disclosed herein, in part, are reagent cycling sequencing methods,wherein sequencing reagents are introduced, one after another, for everycycle of examination and/or incorporation. Optionally, the sequencingreaction mixture includes a polymerase, a primed template nucleic acid,and at least one type of nucleotide. Optionally, the nucleotide and/orpolymerase are introduced cyclically to the sequencing reaction mixture.Optionally, the sequencing reaction mixture includes a plurality ofpolymerases, primed template nucleic acids, and nucleotides. Optionally,a plurality of nucleotides and/or a plurality of polymerases areintroduced cyclically to the sequencing reaction mixture. Optionally,the examination step of the sequencing reaction has a differentcomposition than the incorporation step of the sequencing reaction.

Optionally, one or more nucleotides are sequentially added to andremoved from the sequencing reaction. Optionally, 1, 2, 3, 4, or moretypes of nucleotides are added to and removed from the reaction mixture.For example, one type of nucleotide is added to the sequencing reaction,removed, and replaced by another type of nucleotide. Optionally, anucleotide type present during the examination step is different from anucleotide type present during the incorporation step. Optionally, anucleotide type present during one examination step is different from anucleotide type present during a sequential examination step (i.e., thesequential examination step is performed prior to an incorporationstep). Optionally, 1, 2, 3, 4 or more types of nucleotides are presentin the examination reaction mixture and 1, 2, 3, 4, or more types ofnucleotides are present in the incorporation reaction mixture.

Optionally, a crippled DNA polymerase is cyclically added to and removedfrom the sequencing reaction. One or more different types of polymerasesmay be cyclically added to and removed from the sequencing reaction.Optionally, a polymerase type present during the examination step isdifferent from a polymerase type present during the incorporation step.A polymerase type present during one examination step may be differentfrom a polymerase type present during a sequential examination step(i.e., the sequential examination step is performed prior to anincorporation step).

Optionally, conditions such as the presence of reagents, pH,temperature, and ionic strength are varied throughout the sequencingreaction. Optionally, a metal is cyclically added to and removed fromthe sequencing reaction. For example, a catalytic metal ion may beabsent during an examination step and present during an incorporationstep. Alternatively, a polymerase inhibitor may be present during anexamination step and absent during an incorporation step. Optionally,reaction components that are consumed during the sequencing reaction aresupplemented with the addition of new components at any point during thesequencing reaction.

Nucleotides can be added one type at a time, with the crippled DNApolymerase, to a reaction condition that favors closed-complexformation. The polymerase binds only to the template nucleic acid if thenext correct nucleotide is present. A wash step after every nucleotideaddition ensures all excess polymerases and nucleotides not involved ina closed-complex are removed from the reaction mixture. If thenucleotides are added one at a time, in a known order, the next base onthe template nucleic acid is determined by the formation of aclosed-complex when the added nucleotide is the next correct nucleotide.The closed-complex may be identified by both the conformational changeand the increased stability of the polymerase-template nucleicacid-nucleotide interaction. Optionally, the stability of theclosed-complex formed in the presence of the next correct nucleotide isat least an order of magnitude greater than the unstable interactions ofthe polymerase with the template nucleic acid in the presence ofincorrect nucleotides. The use of a wash step ensures that there are nounbound nucleotides and polymerases and that the only nucleotidespresent in the reaction are those sequestered in a closed-complex with apolymerase and a template nucleic acid. Once the next base on thetemplate nucleic acid is determined, the next correct nucleotidesequestered in the closed-complex may be incorporated by flowing inreaction conditions that favor dissociation or destabilization of theclosed-complex and extending the template nucleic acid primer strand byone base (incorporation). Therefore, the wash step ensures that the onlynucleotide incorporated is the next correct nucleotide from theclosed-complex. This reagent cycling method may be repeated and thenucleic acid sequence determined. This reagent cycling method may beapplied to a single template nucleic acid molecule, or to collections ofclonal populations such as PCR products or rolling-circle amplified DNA.Many different templates can be sequenced in parallel if they arearrayed, for instance, on a solid support. Optionally, the wash stepdestabilizes binary complex formation. Optionally, the washing isperformed for a duration of time that ensures that the binary complex isremoved, leaving the stabilized closed-complex in the reaction mixture.Optionally, the wash step includes washing the reaction with a highionic strength or a high pH solution.

Optionally, the incorporation step is a three stage process. In thefirst stage, all four nucleotide types are introduced into a reactionincluding a primed template nucleic acid, with a crippled DNApolymerase, under reaction conditions which favor the formation of aclosed-complex, and the next correct nucleotides are allowed to formstable closed-complexes with the template nucleic acid. In a secondstage, the crippled DNA polymerase and any cognate nucleotide that mayhave been present is removed, and the removed components are thenreplaced with a second polymerase and one or more nucleotides (e.g.,reversible terminator nucleotides). In a third stage, cognate nucleotideis incorporated into the 3′-end of the template nucleic acid primer.Formation of tight polymerase-nucleic acid complexes in theincorporation step can be enabled by standard techniques such as fusinga non-specific DNA binding domain to the polymerase (e.g., the Phusionpolymerase, which is available from Thermo Fisher Scientific; Waltham,Mass.), and utilizing high concentrations of nucleotides to ensurecorrect nucleotides are always present in the closed-complex.

Polymerase molecules bind to primed template nucleic acid molecules in afingers-closed conformation in the presence of the next correctnucleotide even in the absence of divalent metal ions that are typicallyrequired for polymerase synthesis reactions. The conformational changetraps the nucleotide complementary to the next template base within theactive site of the polymerase. Optionally, the formation of theclosed-complex may be used to determine the identity of next base on thetemplate nucleic acid. Optionally, the primed template nucleic acids maybe contacted serially by different nucleotides in the presence ofpolymerase, in the absence of catalytic divalent metal ions; wherein theformation of a closed-complex indicates the nucleotide currently incontact with the template nucleic acid is the complementary nucleotideto the next base on the nucleic acid. A known order of nucleotides (inthe presence of polymerase and absence of catalytic metal ions) broughtinto contact with the template nucleic acid ensures facileidentification of the complementary nucleotide based on the particularposition in the order that induces closed-complex formation. Optionally,an appropriate wash step may be performed after every nucleotideaddition to ensure removal of all excess enzymes and nucleotides,leaving behind only the polymerase that is bound to nucleic acids in aclosed-complex with the next correct nucleotide at the active site. Theclosed-complex may be identified by means that reveal the conformationalchange of the polymerase in the closed conformation or by means thatreveal the increased stability of thepolymerase/nucleic-acid/next-correct-nucleotide complex compared tobinary polymerase-nucleic acid complexes or compared to unstableinteractions between the polymerase, primed template nucleic acid andincorrect nucleotides.

Optionally, the process of identifying the next complementary nucleotide(examination step) includes the steps of contacting immobilized primedtemplate nucleic acids with an examination mixture including polymeraseand nucleotides of one kind under conditions that inhibit the chemicalincorporation of the nucleotide, removing unbound reagents by a washstep, detecting the presence or absence of polymerase closed-complex onthe immobilized nucleic acids, and repeating these steps serially, withnucleotides of different kinds until a closed-complex formation isdetected. The closed-complex may be identified by both theconformational change and the increased stability of thepolymerase/nucleic acid/next-correct-nucleotide complex. The wash stepbetween successive nucleotide additions may be eliminated by the use ofdetection mechanisms that can detect the formation of the closed-complexwith high fidelity, for instance, evanescent wave sensing methods ormethods that selectively monitor signals from the closed-complex. Theexamination steps noted above may be followed by an incorporation stepincluding, contacting the closed-complex with catalytic metal ions(e.g., Mn²⁺) to covalently add the nucleotide sequestered in theclosed-complex to the 3′-end of the primer. Optionally, theincorporation step may include, contacting the immobilized nucleic acidswith a pre-incorporation mixture including a combination of multipletypes of nucleotides and polymerase under conditions that inhibit thechemical incorporation of the nucleotides; wherein the pre-incorporationmixture may contain additives and solution conditions to ensure highlyefficient closed-complex formation (e.g., low-salt conditions). Themethods may also include performing a wash step to remove unboundreagents and providing the immobilized complexes with an incorporationmixture, including catalytic metal ions, to chemically incorporatenucleotides sequestered within the active site of the polymerase. Thepre-incorporation mixture ensures highly efficient closed-complexformation, while the wash step and incorporation mixture ensure theaddition of a single nucleotide to the 3′-end of the primer. Optionally,the incorporation step may occur directly after examination an additionof one type of nucleotide. For instance, a repeated pattern used forsequencing may include the following flow pattern (i) dATP+/polymerase,(ii) Wash, (iii) Mn⁺², (iv) Wash, (v) dTTP+/polymerase, (vi) Wash, (vii)Mn⁺², (viii) Wash, (ix) dCTP+/polymerase, (x) Wash (xi) Mn⁺², (xii)Wash, (xiii) dGTP+/polymerase, (xiv) Wash, (xv) Mn⁺², (xvi) Wash.Optionally, the repeated pattern used for sequencing may include (i)dATP+/polymerase, (ii) Wash, (iii) dTTP+/polymerase, (iv) Wash, (v)dGTP+/polymerase, (vi) Wash, (vii) dCTP+/polymerase, (viii) Wash, (ix)Pre-incorporation mixture, (x) Wash, (xi) Mn²⁺, (xii) Wash. The washsteps optionally contain metal ion chelators and other small moleculesto prevent accidental incorporations during the examination steps. Afterthe incorporation step, the primer strand is typically extended by onebase. Repeating this process, sequential nucleobases of a nucleic acidmay be identified, effectively determining the nucleic acid sequence.Optionally, the examination step is performed at high salt conditions,for example, under conditions of 50 mM to 1,500 mM salt.

For sequencing applications, it can be advantageous to minimize oreliminate fluidics and reagents exchange. Removing pumps, valves andreagent containers can allow for simplified manufacturing of smallerdevices. Disclosed herein, in part, are “all-in” sequencing methods,wherein the need to introduce reagents one after another, for everycycle of examination and/or incorporation, is eliminated. Reagents areadded only once to the reaction, and sequencing-by-synthesis isperformed by manipulating reagents already enclosed within thesequencing reaction. A scheme such as this requires a method todistinguish different nucleotides, a method to synchronize incorporationof nucleotides across a clonal population of nucleic acids and/or acrossdifferent nucleic acid molecules, and a method to ensure only onenucleotide is added per cycle.

Optionally, the sequencing reaction mixture includes a crippled DNApolymerase, a primed template nucleic acid, and at least one type ofnucleotide. Optionally, the sequencing reaction mixture includes aplurality of polymerases, primed template nucleic acids, andnucleotides. As provided herein, a polymerase refers to a singlepolymerase or a plurality of polymerases. As provided herein, a primedtemplate nucleic acid or template nucleic acid refers to a single primedtemplate nucleic acid or single template nucleic acid, or a plurality ofprimed template nucleic acids or a plurality of template nucleic acids.As provided herein, a nucleotide refers to one nucleotide or a pluralityof nucleotides. As provided herein, a single nucleotide is onenucleotide. Optionally, the sequencing reaction nucleotides include, butare not limited to, 1, 2, 3, or 4 of the following nucleotides: dATP,dGTP, dCTP, dTTP, and dUTP.

Optionally, 1, 2, 3, 4 or more types of nucleotides (e.g., dATP, dGTP,dCTP, dTTP) are present in the reaction mixture together at the sametime, wherein one type of nucleotide is a next correct nucleotide. Thereaction mixture further includes at least one crippled DNA polymeraseand at least one primed template nucleic acids. Optionally, the templatenucleic acid is a clonal population of template nucleic acids.Optionally, the crippled DNA polymerase, primed template nucleic acid,and the nucleotide form a closed-complex under examination reactionconditions.

In the provided methods, four types of nucleotides can be present atdistinct and different concentrations wherein the diffusion and bindingtimes of the polymerase to the template nucleic acid are different foreach of the four nucleotides, should they be the next correctnucleotide, due to the different concentrations of the four nucleotides.For example, the nucleotide at the highest concentration would bind toits complementary base on the template nucleic acid at a fast time, andthe nucleotide at the lowest concentration would bind to itscomplementary base on the template nucleic acid at a slower time;wherein binding to the complementary base on the template nucleic acidrefers to the polymerase binding to the template nucleic acid with thenext correct nucleotide in a closed closed-complex. The identity of thenext correct nucleotide is therefore determined by monitoring the rateor time of binding of polymerase to the template nucleic acid in aclosed-complex. Optionally, the four types of nucleotides may bedistinguished by their concentration, wherein the differentconcentrations of the nucleotides result in measurably differenton-rates for the polymerase binding to the nucleic acid. Optionally, thefour types of nucleotides may be distinguished by their concentration,wherein the different concentrations of the nucleotides result inmeasurably different on-rates for the formation of a stabilizedclosed-complex.

Optionally, the crippled DNA polymerase is labeled. In some instances,the polymerase is not labeled (i.e., does not harbor an exogenous label,such as a fluorescent label) and any label-free detection methoddisclosed herein or known in the art is employed. Optionally, thebinding of the polymerase to the nucleic acid is monitored via adetectable feature of the crippled DNA polymerase. Optionally, theformation of a stabilized closed-complex is monitored via a detectablefeature of the polymerase. A detectable feature of the polymerase mayinclude, but is not limited to, optical, electrical, thermal,colorimetric, mass, and any combination thereof.

Optionally, 1, 2, 3, 4, or more nucleotides types (e.g., dATP, dTTP,dCTP, dGTP) are tethered to 1, 2, 3, 4, or more different crippled DNApolymerase; wherein each nucleotide type is tethered to a differentpolymerase and each polymerase has a different exogenous label or adetectable feature from the other polymerases to enable itsidentification. All tethered nucleotide types can be added together to asequencing reaction mixture forming a closed-complex including atethered nucleotide-polymerase; the closed-complex is monitored toidentify the polymerase, thereby identifying the next correct nucleotideto which the polymerase is tethered. The tethering may occur at thegamma phosphate of the nucleotide through a multi-phosphate group and alinker molecule. Such gamma-phosphate linking methods are standard inthe art, where a fluorophore is attached to the gamma phosphate linker.Optionally, different nucleotide types are identified by distinguishableexogenous labels. Optionally, the distinguishable exogenous labels areattached to the gamma phosphate position of each nucleotide.

Optionally, the sequencing reaction mixture includes a catalytic metalion. Optionally, the catalytic metal ion is Mn²⁺ ion. Optionally, thecatalytic metal ion is available to react with a crippled DNA polymeraseat any point in the sequencing reaction in a transient manner. To ensurerobust sequencing, the catalytic metal ion is available for a briefperiod of time, allowing for a single nucleotide complementary to thenext base in the template nucleic acid to be incorporated into the3′-end of the primer during an incorporation step. In this instance, noother nucleotides, for example, the nucleotides complementary to thebases downstream of the next base in the template nucleic acid, areincorporated. Optionally, the catalytic metal ion magnesium is presentas a photocaged complex (e.g., DM-Nitrophen) in the sequencing reactionmixture such that localized UV illumination releases the magnesium,making it available to the polymerase for nucleotide incorporation.Furthermore, the sequencing reaction mixture may contain EDTA, whereinthe magnesium is released from the polymerase active site aftercatalytic nucleotide incorporation and captured by the EDTA in thesequencing reaction mixture, thereby rendering magnesium incapable ofcatalyzing a subsequent nucleotide incorporation.

Thus, in the provided methods, a catalytic metal ion can be present in asequencing reaction in a chelated or caged form from which it can bereleased by a trigger. For example, the catalytic metal ion catalyzesthe incorporation of the closed-complex next correct nucleotide, and, asthe catalytic metal ion is released from the active site, it issequestered by a second chelating or caging agent, disabling the metalion from catalyzing a subsequent incorporation. The localized release ofthe catalytic metal ion from its cheating or caged complex is ensured byusing a localized uncaging or un-chelating scheme, such as an evanescentwave illumination or a structured illumination. Controlled release ofthe catalytic metal ions may occur for example, by thermal means.Controlled release of the catalytic metal ions from their photocagedcomplex may be released locally near the template nucleic acid byconfined optical fields, for instance by evanescent illumination such aswaveguides or total internal reflection microscopy. Controlled releaseof the catalytic metal ions may occur for example, by altering the pH ofthe solution near the vicinity of the template nucleic acid. Chelatingagents such as EDTA and EGTA are pH dependent. At a pH below 5, divalentcations Mg²⁺ and Mn²⁺ are not effectively chelated by EDTA. A method tocontrollably manipulate the pH near the template nucleic acid allows thecontrolled release of a catalytic metal ion from a chelating agent.Optionally, the local pH change is induced by applying a voltage to thesurface to which the nucleic acid is attached. The pH method offers anadvantage in that that metal goes back to its chelated form when the pHis reverted back to the chelating range.

Described above are polymerase-nucleic acid binding reactions for theidentification of a nucleic acid sequence. However, nucleic acidsequence identification may include information regarding nucleic acidmodifications, including methylation and hydroxymethylation. Methylationmay occur on cytosine bases of a template nucleic acid. DNA methylationmay stably alter the expression of genes. DNA methylation is alsoindicated in the development of various types of cancers,atherosclerosis, and aging. DNA methylation therefore can serve as anepigenetic biomarker for human disease.

Optionally, one or more cytosine methylations on a template nucleic acidare identified during the sequencing by binding methods provided herein.The template nucleic acid may be clonally amplified prior to sequencing,wherein the amplicons include the same methylation as their templatenucleic acid. Amplification of the template nucleic acids may includethe use of DNA methyltransferases to achieve amplicon methylation. Thetemplate nucleic acids or amplified template nucleic acids are providedto a reaction mixture including a polymerase and one or more nucleotidetypes, wherein the interaction between the polymerase and nucleic acidsis monitored. Optionally, the interaction between the polymerase andtemplate nucleic acid in the presence of a methylated cytosine isdifferent than the interaction in the presence of an unmodifiedcytosine. Therefore, based on examination of a polymerase-nucleic acidinteraction, the identity of a modified nucleotide is determined.

Optionally, following one or more examination and/or incorporationsteps, a subset of nucleotides is added to reduce or reset phasing.Thus, the methods can include one or more steps of contacting a templatenucleic acid molecule being sequenced with a composition comprising asubset of nucleotides and an enzyme for incorporating the nucleotidesinto the strand opposite the template strand of the nucleic acidmolecule. The contacting can occur under conditions to reduce phasing inthe nucleic acid molecule. Optionally, the step of contacting thetemplate nucleic acid molecule occurs after an incorporation step and/orafter an examination step. Optionally, the contacting occurs after 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75,80, 85, 90, 95, or 100 rounds or more of sequencing (i.e., rounds ofexamination and incorporation). Optionally, the contacting occurs after30 to 60 rounds of sequencing. Optionally, the contacting occurs afterevery round of sequencing (i.e., after one set of examination andincorporation steps). Optionally, multiple contacting steps occur afterevery round of sequencing, wherein each contacting step may comprisedifferent subsets of nucleotides. Optionally, the method furthercomprises one or more washing steps after contacting. Optionally, thesubset comprises two or three nucleotides. Optionally, the subsetcomprises three nucleotides. Optionally, the subset of nucleotides isselected from three of dATP, dGTP, dCTP, dTTP or a derivative thereof.Optionally, the three nucleotides comprise adenosine, cytosine, andguanine. Optionally, the three nucleotides comprise adenosine, cytosine,and thymine. Optionally, the three nucleotides comprise cytosine,guanine and thymine. Optionally, the three nucleotides compriseadenosine, guanine and thymine. Optionally, each round of contactingcomprises the same subset or different subsets of nucleotides.Optionally, sequencing of a nucleic acid template is monitored and thecontacting with the subset of nucleotides occurs upon detection ofphasing. See also for example, U.S. Pat. No. 8,236,532, which isincorporated herein by reference in its entirety.

Optionally, the sequencing reaction involves a plurality of templatenucleic acids, polymerases and/or nucleotides, wherein a plurality ofclosed-complexes is monitored. Clonally amplified template nucleic acidsmay be sequenced together wherein the clones are localized in closeproximity to allow for enhanced monitoring during sequencing.Optionally, the formation of a closed-complex ensures the synchronicityof base extension across a plurality of clonally amplified templatenucleic acids. The synchronicity of base extension allows for theaddition of only one base per sequencing cycle.

Genotyping Applications

Exemplary uses for the disclosed crippled DNA polymerases relate to SNPdetection and genotyping applications, without necessitating extensivesequence determination. Here the crippled DNA polymerase can be used fordetermining, without incorporation, the identity of the next correctnucleotide for a primed template nucleic acid molecule undergoingexamination. Optionally, the procedure involves hybridizing a primer toa target nucleic acid to result in the primed template nucleic acidmolecule. The primed template nucleic acid can then be contacted with acomposition that includes the crippled DNA polymerase and one of morenucleotides. Optionally, the crippled DNA polymerase includes adetectable label, such as a fluorescent detectable label. Optionally,more than one crippled DNA polymerase is included in the composition,where at least two of the crippled DNA polymerases are distinguishablylabeled. Optionally, the composition includes at least one nucleotidethat harbors a detectable label, such as a detectable fluorescent label.Optionally, more than one nucleotide in the composition includes adetectable label. Optionally, two different nucleotides in thecomposition each harbor different detectable labels that can bedistinguished from each other. Commonly owned U.S. patent applicationidentified by Ser. No. 62/448,630 (filed Jan. 20, 2017), the disclosureof which is incorporated by reference, sets forth numerous genotypingprocedures wherein the instant crippled DNA polymerases can be used.

Illustrative Sequencing Methods Employing a Crippled DNA Polymerase

Examination reactions in accordance with the disclosed techniqueoptionally can be conducted in the presence of Mg⁺² ion. The fact thatthe crippled DNA polymerase cannot catalyze phosphodiester bondformation in the presence of this ion means that it can be includedduring the examination step without compromising transient binding(i.e., binding without incorporation). The presence of the catalyticmetal ions may enhance the discriminatory activities that are ordinarilyimportant during the DNA polymerization reaction. While non-catalyticions also may be included or substituted in place of the catalytic ions,inclusion of the non-catalytic metal ions that inhibitpolymerase-mediated incorporation is optional.

Examination reactions using the disclosed technique optionally can beconducted using native nucleotides. Generally speaking, any method fordetecting a ternary complex that includes the crippled DNA polymerasewill be useful in the nucleotide sequence determination protocol.Optical techniques detecting changes in refractive index (e.g.,interferometry or surface plasmon resonance sensing) will be capable oflabel-free detection, and so can be carried out using unlabeled nativenucleotides in the examination step. Optionally, if the crippled DNApolymerase harbors an exogenous label (e.g., a fluorescent label), thenformation of a ternary complex localized to a defined position on asolid support or surface (e.g., a locus on a planar array, or a beadarray) can be detected using conventional fluorescent monitoringtechniques.

Examination reactions conducted using crippled polymerases optionallycan be carried out using nucleotides that harbor an exogenous label(e.g., a fluorescent label). When used with a fluorescent detectionplatform, nucleotide analogs harboring fluorescent chemical moietiesthat co-localize with the primed template nucleic acid and the crippledpolymerase can indicate ternary complex formation.

Examination can be conducted using primed template nucleic acids, wherethe primer strand of the duplex harbors a free 3′ hydroxyl moiety thatis available to participate in formation of a phosphodiester bond in thepresence of a cognate nucleotide and a native DNA polymerase having thecapability of forming phosphodiester bonds.

An exemplary work flow for the sequencing technique would involve firstcontacting a primed template nucleic acid with one or more testnucleotides (i.e., nucleotides being tested as cognate nucleotidecandidates), and the crippled polymerase. In the event the testnucleotide is the next correct nucleotide, a ternary complex thatincludes all three components will be formed. Affirmative detection ofthe ternary complex indicates that the test nucleotide is the cognatenucleotide harboring a base complementary to that position along thetemplate strand. Since the crippled DNA polymerase will be incapable ofcatalyzing a phosphodiester bond, identification of the next correctnucleotide necessarily will take place without incorporation of thecognate nucleotide into the primer. Extending the primer byincorporation of the cognate nucleotide can be accomplished by differentapproaches. For example, once information needed to identify the nextcorrect nucleotide has been gathered, an exchange of reaction mixturescan be effected so that the crippled polymerase is exchanged for asecond polymerase able to promote phosphodiester bond formation. If alsoprovided with cognate nucleotide and a divalent cation, the secondpolymerase will be able to effect incorporation of the cognatenucleotide into the primer.

It is to be understood that the disclosed technique will require anactive DNA polymerizing enzyme for incorporating cognate nucleotide intothe growing primer.

In certain embodiments of the disclosed technique, a reversibleterminator nucleotide corresponding to the cognate nucleotide can beincorporated into the growing primer once information needed toestablish identity of the cognate nucleotide has been gathered.

In one aspect, the disclosed technique features nucleotideidentification by a procedure that relies on use of a crippled DNApolymerase. Optionally, Mg²⁺ ions can be included in the examinationreaction mixture that further includes the primed template nucleic acid,crippled DNA polymerase, and test nucleotide. Optionally, the primedtemplate nucleic acid can be immobilized at a fixed position on a planarsurface or a bead. Optionally, the test nucleotides used in theexamination step are native nucleotides. Optionally, the testnucleotides include an exogenous label, such as a fluorescent label.Optionally, different test nucleotides are labeled with differentexogenous labels that are distinguishable from each other. Optionally,the primer of the primed template nucleic acid includes a free 3′hydroxyl group. Optionally, the crippled DNA polymerase harbors anexogenous label, such as a fluorescent label. Optionally, oncesufficient information has been gathered to identify a cognatenucleotide present in a ternary complex that includes the primedtemplate nucleic acid, the crippled DNA polymerase, and the nucleotide,the crippled DNA polymerase and the nucleotide can be removed from theprimed template nucleic acid (e.g., by the use of EDTA and high ionicstrength conditions) and replaced with a second polymerase and one oranother type of nucleotide. The second DNA polymerase optionally will becapable of catalyzing phosphodiester bond formation. Nucleotides used inconjunction with the second DNA polymerase can be either nativenucleotides or nucleotide analogs (e.g., reversible terminatornucleotides). Optionally, using the crippled DNA polymerase and thesecond DNA polymerase in a cycling protocol permits extension of theprimer by incorporation of one or more nucleotides. Optionally, use ofthe second DNA polymerase in combination with reversible terminatornucleotides restricts the incorporation to a single nucleotide.Optionally, the reversible terminator moiety can be removed to permitsubsequent incorporation of another nucleotide or nucleotide analog.

Example 1 illustrates how a crippled polymerase can be used in a DNAsequencing method with fluorescently labeled nucleotides.

Example 1 DNA Sequence Determination Using a Crippled DNA Polymerase andFluorescent Nucleotides

A crippled DNA polymerase possessing cognate nucleotide binding anddiscrimination activities, but not the ability to form phosphodiesterbonds first is obtained. A primed template nucleic acid immobilized at adefined position on a solid support in an array format of a flow cell iscontacted with a first reaction mixture that includes the crippledpolymerase, and dATP labeled with a fluorescent moiety on the gammaphosphate. Fluorescence monitoring indicates no fluorescence signalabove background. This indicates that dATP is not the next correctnucleotide. The first reaction mixture within the flow cell is replacedby a second reaction mixture that includes the crippled polymerase, anddGTP labeled with a fluorescent moiety on the gamma phosphate.Fluorescence monitoring indicates substantial fluorescence abovebackground. This indicates that dGTP is the next correct nucleotide. Thesecond reaction mixture is flushed from the flow cell and replaced by anincorporation reaction mixture that includes Therminator DNA polymerase(New England BioLabs) and four reversible terminator nucleotides, eachhaving a 3′-ONH₂ blocking group. The reversible terminator nucleotidehaving guanine as its base is incorporated. Optionally, the resultingblocked primed template nucleic acid molecule is used directly in asubsequent round of nucleotide examination with the crippled DNApolymerase to obtain more extensive sequence information. Optionally,the reversible terminator moiety is chemically cleaved by standardprocedures to reveal a native primer, and the resulting primed templatenucleic acid molecule is used in a subsequent round of nucleotideexamination with the crippled DNA polymerase to obtain more extensivesequence information.

Example 2 illustrates how a crippled polymerase can be used in a DNAsequencing method when the crippled polymerase is fluorescently labeled.

Example 2 DNA Sequence Determination Using a Fluorescently LabeledCrippled DNA Polymerase

A crippled DNA polymerase possessing cognate nucleotide binding anddiscrimination activities, but not the ability to form phosphodiesterbonds first is obtained. The crippled DNA polymerase is labeled with afluorescent moiety on the functional group of a surface-accessiblecysteine amino acid. A primed template nucleic acid immobilized at adefined position on a solid support in an array format of a flow cell iscontacted with a first reaction mixture that includes the fluorescentcrippled DNA polymerase, and native dATP. Fluorescence monitoringindicates no fluorescence signal above background. This indicates thatdATP is not the next correct nucleotide. The first reaction mixturewithin the flow cell is replaced by a second reaction mixture thatincludes the fluorescent crippled polymerase, and native dGTP.Fluorescence monitoring indicates substantial fluorescence abovebackground. This indicates that dGTP is the next correct nucleotide. Thesecond reaction mixture is flushed from the flow cell and replaced by anincorporation reaction mixture that includes Therminator DNA polymerase(New England BioLabs) and four reversible terminator nucleotides, eachhaving a 3′-ONH₂ blocking group. The reversible terminator nucleotidehaving guanine as its base is incorporated.

The above-described TDE mutant polymerase was prepared and purifiedusing standard techniques that will be familiar to those having anordinary level of skill in the art. The Bst-f protein backbone used inthe procedure (i.e., SEQ ID NO:1) had been modified to include anoptimized cysteine (cys) residue at position 23, a histidine-tagsequence at the N-terminus (i.e., positions 5-10) to aid in proteinpurification, and a thrombin cleave site between positions 16-17. Noneof the protein sequence modifications upstream of the first methionineof the native Bst DNA polymerase (i.e., position 27 of SEQ ID NO:1) wasdeemed essential for the desired combination of correct nucleotideidentification without Mg²⁺-catalyzed incorporation. Thus, inclusion ofthese modifications is optional in the working product, and so the threeTDE mutants set forth in Table 1 (i.e., using the parent scaffolds ofany of SEQ ID Nos:1-3) optionally may be used for carrying out detectionor sequencing procedures.

Example 3 describes the use of crippled DNA polymerases insequencing-by-binding protocols involving cycles of examination toidentify cognate nucleotides, and demonstrates that the mutants do notincorporate cognate nucleotide in the presence of the otherwisecatalytic Mg²⁺ metal ion. As described above, the TDE mutant includes asingle amino acid change at position 381 of the Bst-f enzyme (SEQ IDNO:1). Accordingly, the TDE mutant included the sequence EYSQIELR (SEQID NO:12) in place of DYSQIELR (SEQ ID NO:13) within motif A. The BDEmutant included the sequence QVHEEL (SEQ ID NO:14) in place of QVHDEL(SEQ ID NO:15) within motif C. The parent enzyme that included anexogenous cysteine residue and N-terminal His-tag, but did not includeany polymerization-crippling mutation served as a control in thisprocedure. In alternatives, engineered polymerases including themodified motif A can be substituted in the procedure. For example, theengineered polymerase can include the sequence of SEQ ID NO:12 containedwithin the sequence of SEQ ID NO:3 having position 355 substituted byglutamate. The sequence of SEQ ID NO:2 having position 364 substitutedby glutamate is one example, while the sequence of SEQ ID NO:1 havingposition 381 substituted by glutamate is another example. Likewise,engineered polymerases including the modified motif C can be substitutedin the procedure. For example, the engineered polymerase can include thesequence of SEQ ID NO:14 contained within the sequence of SEQ ID NO:3having position 532 substituted by glutamate. The sequence of SEQ IDNO:2 having position 541 substituted by glutamate is one example, whilethe sequence of SEQ ID NO:1 having position 558 substituted by glutamateis another example.

Example 3 Demonstration of Cognate Nucleotide Identification withoutIncorporation Using a Crippled DNA Polymerase

A FORTEBIO® (Menlo Park, Calif.) Octet instrument employing biolayerinterferometry to measure binding reactions at the surface of a fiberoptic tip was used in a multiwell plate format to illustrate thesequencing technique. Primed template nucleic acid moleculesbiotinylated at the 5′-ends of the template strand were immobilized ontofiber optic tips functionalized with streptavidin (SA) using standardprocedures. The primed template nucleic acid molecule in this procedurehad CG as the next correct nucleotides downstream of the primer.

The cycling procedure involved steps for: (1) washing/regeneratingsensor tips; (2) forming ternary complexes that included polymerase andcognate nucleotides; and (3) washing with an EDTA solution to stripcomplexes from the primed template nucleic acid molecule. Anincorporation step followed a complete round of binding and examinationusing the four dNTPs, one at a time. Sensor tips were washed/regeneratedin a Tris-buffered solution (pH 8.0) that included KCl, potassiumglutamate, and 0.01% Tween-20 before commencing the cycling protocol.The first incoming nucleotide was interrogated with 500 nM of either TDEor 500 nM of CBT in the presence of examination buffer (30 mM Tris-HCl(pH 8.0), 420 mM KCl, 160 mM potassium glutamate, 2 mM SrCl₂, 0.01%Tween-20, 0.1 mg/mL acetylated BSA, and 1 mM β-mercaptoethanol. Nativenucleotides were employed in the procedure, and were contacted to thesensor tip in the following order: dATP, dCTP, dTTP, and dGTP. Each ofthe dNTPs was present at a concentration of 100 μM, except for dTTP,which was used at a concentration of 200 μM. Nucleotide binding stepswere for a period of about 30 seconds at 30° C. At the end of eachnucleotide binding and examination step, any formed complexes werewashed from the sensor tip for 45 seconds using an EDTA solutioncontaining KCl to chelate divalent cations. Thereafter, the biosensorwas regenerated for 30 seconds before moving to the next dNTP exam.

Following examination of all four dNTPs to determine whether a ternarycomplex had formed, incorporation reactions were performed toinvestigate possible residual polymerase activity of the TDE mutant.Here the parent CBT enzyme served as a positive control for nucleotidebinding and incorporation activities. First, ternary complexes wereprepared by contacting the sensor tips with the cognate nucleotide(i.e., dCTP) at a concentration of 100 μM for 30 seconds. Next,biosensor tips were transferred to an incorporation buffer (30 mMTris-HCl (pH 8.0), 50 mM KCl, 50 mM Mg²⁺) for 30 seconds. Finally,complexes were washed from the sensor tips for 45 seconds using the EDTAsolution containing KCl to chelate divalent cations. Again, thebiosensor was regenerated for 30 seconds before moving to the nextseries of examination reactions using all four dNTPs, one at a time.Results from this latter set of examination reactions was informativeregarding binding and incorporation activities of the mutant enzyme.

Results from the procedure, shown in FIG. 1, confirmed that the crippledpolymerase correctly identified the cognate nucleotide, but wasincapable of incorporating that nucleotide even in the presence of thecatalytic Mg²⁺ metal ion. The FIGURE shows examination traces for allfour nucleotides conducted using the TDE and CBT polymerases. Formationof binary complexes before addition of nucleotide established baselinevalues for the comparison. Ternary complexes generated in the presenceof dNTP indicated that both polymerases correctly identified dCTP as thecognate nucleotide. In all cases, non-cognate nucleotides wereassociated with substantially baseline binding signals. Following thestep to permit incorporation, only the parent CBT enzyme was shown topossess catalytic activity. More specifically, the mutant TDE enzymeagain identified dCTP as the cognate nucleotide for the primed templatenucleic acid molecule. This indicated that no nucleotide had beenincorporated by the mutant enzyme under incorporating conditions.Conversely, the parent CBT enzyme identified dGTP as the next correctnucleotide following incorporation. Thus, the crippled polymerasecorrectly performed the examination step without the ability toincorporate cognate nucleotide. Of course, a repetitive cyclingprocedure to conduct extensive sequence determination can use adifferent enzyme for the incorporation step. A reversible terminatornucleotide (e.g., an unlabeled reversible terminator nucleotide) may beused in the incorporation procedure.

Additional testing conducted at lower concentrations of catalytic metalions was performed to investigate the ability of the crippled TDEpolymerase to incorporate cognate nucleotides by phosphodiester bondformation. Here 10 mM concentrations of MgCl₂ or MnCl₂ were tested inthe incorporation step. Again, the crippled TDE mutant failed toincorporate cognate nucleotide in the presence of the catalytic Mg²⁺metal ion. However, the TDE mutant incorporated cognate nucleotide inthe presence of the catalytic Mn²⁺ metal ion. Significantly, paralleltesting showed that the BDE mutant correctly identified cognatenucleotide during examination steps, but like the TDE mutant also wascatalytically inactive in the presence of Mg²⁺ ion. The TDN mutant wasincapable of both identifying cognate nucleotide and catalyzingphosphodiester bond formation.

Disclosed above are materials, compositions, and components that can beused for, can be used in conjunction with, can be used in preparationfor, or are products of the disclosed methods and compositions. It is tobe understood that when combinations, subsets, interactions, groups,etc. of these materials are disclosed, and that while specific referenceof each various individual and collective combinations and permutationsof these compounds may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a method is disclosedand discussed and a number of modifications that can be made to a numberof molecules including the method are discussed, each and everycombination and permutation of the method, and the modifications thatare possible are specifically contemplated unless specifically indicatedto the contrary. Likewise, any subset or combination of these is alsospecifically contemplated and disclosed. This concept applies to allaspects of this disclosure, including steps in methods using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed, it is understood that each of these additionalsteps can be performed with any specific method steps or combination ofmethod steps of the disclosed methods, and that each such combination orsubset of combinations is specifically contemplated and should beconsidered disclosed.

Publications cited herein, and the material for which they are cited,are hereby specifically incorporated by reference in their entireties.All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

It is to be understood that the headings used herein are fororganizational purposes only and are not meant to limit the descriptionor claims.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the claims.

What is claimed is:
 1. A method of determining whether a test nucleotideis the next correct nucleotide comprising a base complementary to thenext base in a template strand immediately downstream of a primer in aprimed template nucleic acid, comprising the steps of: (a) contactingthe primed template nucleic acid with a first reaction mixture thatcomprises a crippled DNA polymerase comprising either a polypeptidesequence comprising SEQ ID NO:12 or a polypeptide sequence comprisingSEQ ID NO:14 and the test nucleotide, whereby, if the test nucleotide isthe next correct nucleotide, there is formed a complex comprising theprimed template nucleic acid, the crippled DNA polymerase and the testnucleotide, and wherein the crippled DNA polymerase is substantiallyincapable of magnesium-catalyzed phosphodiester bond formation; (b)measuring binding of the primed template nucleic acid to the crippledDNA polymerase in the presence of the test nucleotide, without chemicalincorporation of the test nucleotide into the primer of the primedtemplate nucleic acid; and (c) determining from the results of step (b)whether the test nucleotide is the next correct nucleotide.
 2. Themethod of claim 1, wherein the crippled DNA polymerase catalyzesformation of phosphodiester bonds in the presence of divalent manganeseions, and wherein the first reaction mixture does not contain aconcentration of divalent manganese ions that promotes formation ofphosphodiester bonds.
 3. The method of claim 1, wherein the testnucleotide comprises an exogenous label.
 4. The method of claim 2,wherein the exogenous label of the test nucleotide comprises afluorescent moiety, and wherein step (b) comprises measuring afluorescent signal produced by the fluorescent moiety of the testnucleotide.
 5. The method of claim 1, wherein the crippled DNApolymerase comprises an exogenous label, and wherein step (b) comprisesdetecting the exogenous label of the crippled DNA polymerase.
 6. Themethod of claim 5, wherein the exogenous label of the crippled DNApolymerase comprises a fluorescent moiety, and wherein step (b)comprises measuring a fluorescent signal produced by the fluorescentmoiety of the crippled DNA polymerase.
 7. The method of claim 1, whereinthe primer comprises a free 3′ hydroxyl moiety.
 8. The method of claim1, further comprising, after step (b), the step of replacing the firstreaction mixture with a second reaction mixture that comprises a secondpolymerase and a second type of nucleotide, and then incorporating thesecond type of nucleotide into the primer of the primed template nucleicacid.
 9. The method of claim 8, wherein the second type of nucleotide isa reversible terminator nucleotide that comprises a reversibleterminator moiety, and wherein incorporation of the reversibleterminator nucleotide produces a blocked primed template nucleic acidmolecule.
 10. The method of claim 9, further comprising the step ofremoving the reversible terminator moiety from the blocked primedtemplate nuclei acid molecule to regenerate the primed template nucleicacid molecule.
 11. The method of claim 9, further comprising repeatingsteps (a)-(c) using the blocked primed template nucleic acid molecule inplace of the primed template nucleic acid.
 12. The method of claim 10,further comprising repeating steps (a)-(c).
 13. The method of claim 1,wherein the polypeptide sequence of the crippled DNA polymerase iseither SEQ ID NO:1 with the exception of comprising SEQ ID NO:12, or SEQID NO:1 with the exception of comprising SEQ ID NO:14.
 14. The method ofclaim 1, wherein the polypeptide sequence of the crippled DNA polymeraseis either SEQ ID NO:2 with the exception of comprising SEQ ID NO:12, orSEQ ID NO:2 with the exception of comprising SEQ ID NO:14.
 15. Themethod of claim 1, wherein the polypeptide sequence of the crippled DNApolymerase is either SEQ ID NO:3 with the exception of comprising SEQ IDNO:12, or SEQ ID NO:3 with the exception of comprising SEQ ID NO:14. 16.The method of claim 1, wherein the first reaction mixture comprisesdivalent magnesium ion.
 17. The method of claim 9, wherein the firstreaction mixture comprises Mg²⁺ ions, and wherein the primer comprises a3′ hydroxyl moiety.